Molecular Antigen Array

ABSTRACT

The present invention is related to the fields of molecular biology, virology, immunology and medicine. The invention provides a composition comprising an ordered and repetitive antigen or antigenic determinant array. The invention also provides a process for producing an antigen or antigenic determinant in an ordered and repetitive array. The ordered and repetitive antigen or antigenic determinant is useful in the production of vaccines for the treatment of infectious diseases, the treatment of allergies and as a pharmaccine to prevent or cure cancer and to efficiently induce self-specific immune responses, in particular antibody responses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/096,970, filed Apr. 28, 2011, now abandoned. U.S. application Ser.No. 13/096,970 is a continuation of U.S. application Ser. No.12/010,107, filed Jan. 18, 2008, now abandoned. U.S. application Ser.No. 12/010,107 is a continuation of U.S. patent Ser. No. 10/050,898,filed Jan. 18, 2002, now U.S. Pat. No. 7,320,793, which claims prioritybenefit of U.S. provisional application No. 60/262,379, filed Jan. 19,2001, U.S. provisional application No. 60/288,549, filed May 4, 2001,U.S. provisional application No. 60/326,998, filed Oct. 5, 2001, andU.S. provisional application No. 60/331,045, filed Nov. 7, 2001. U.S.provisional application No. 60/262,379, filed Jan. 19, 2001, U.S.provisional application No. 60/288,549, filed May 4, 2001, U.S.provisional application No. 60/326,998, filed Oct. 5, 2001, and U.S.provisional application No. 60/331,045, filed Nov. 7, 2001, are herebyincorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to the fields of molecular biology,virology, immunology and medicine. The invention provides a compositioncomprising an ordered and repetitive antigen or antigenic determinantarray. The invention also provides a process for producing an antigen orantigenic determinant in an ordered and repetitive array. The orderedand repetitive antigen or antigenic determinant is useful in theproduction of vaccines for the treatment of infectious diseases, thetreatment of allergies and as a pharmaccine to prevent or cure cancerand to efficiently induce self-specific immune responses, in particularantibody responses.

2. Background Art

WO 00/3227 describes compositions and processes for the production ofordered and repetitive antigen or antigenic determinant arrays. Thecompositions are useful for the production of vaccines for theprevention of infectious diseases, the treatment of allergies and thetreatment of cancers. The compositions comprise a core particle, such asa virus or a virus-like particle, to which at least one antigen or oneantigenic determinant, is associated by way of at least one non-peptidebond leading to the ordered and repetitive antigen array.

Virus-like particles (VLPs) are being exploited in the area of vaccineproduction because of both their structural properties and theirnon-infectious nature. VLPs are supermolecular structures built in asymmetric manner from many protein molecules of one or more types. Theylack the viral genome and, therefore, are noninfectious. VLPs can oftenbe produced in large quantities by heterologous expression and can beeasily be purified.

Examples of VLPs include the capsid proteins of Hepatitis B virus(Ulrich, et al., Virus Res. 50:141-182 (1998)), measles virus (Warnes,et al., Gene 160:173-178 (1995)), Sindbis virus, rotavirus (U.S. Pat.No. 5,071,651 and U.S. Pat. No. 5,374,426), foot-and-mouth-disease virus(Twomey, et al., Vaccine 13:1603-1610, (1995)), Norwalk virus (Jiang,X., et al., Science 250:1580-1583 (1990); Matsui, S. M., et al., J.Clin. Invest. 87:1456-1461 (1991)), the retroviral GAG protein (WO96/30523), the retrotransposon Ty protein p1, the surface protein ofHepatitis B virus (WO 92/11291) and human papilloma virus (WO 98/15631).

It is generally difficult to induce immune responses againstself-molecules due to immunological tolerance. Specifically, lymphocyteswith a specificity for self-molecules are usually hypo- or evenunresponsive if triggered by conventional vaccination strategies.

The amyloid B peptide (Aβ₁₋₄₂) has a central role in the neuropathologyof Alzheimers disease. Region specific, extracellular accumulation of Aβpeptide is accompanied by microgliosis, cytoskeletal changes, dystrophicneuritis and synaptic loss. These pathological alterations are thoughtto be linked to the cognitive decline that defines the disease.

In a mouse model of Alzheimer disease, transgenic animals engineered toproduce Aβ₁₋₄₂ (PDAPP-mice), develop plaques and neuron damage in theirbrains. Recent work has shown immunization of young PDAPP-mice, usingAβ₁₋₄₂, resulted in inhibition of plaque formation and associateddystrophic neuritis (Schenk, D. et al., Nature 400:173-77 (1999)).

Furthermore immunization of older PDAPP mice that had already developedAD-like neuropathologies, reduced the extent and progression of theneuropathologies. The immunization protocol for these studies was asfollows; peptide was dissolved in aqueous buffer and mixed 1:1 withcomplete Freunds adjuvant (for primary dose) to give a peptideconcentration of 100 μg/dose. Subsequent boosts used incomplete Freundsadjuvant. Mice received 11 immunizations over an 11 month period.Antibodies titres greater than 1:10 000 were achieved and maintained.Hence, immunization may be an effective prophylactic and therapeuticaction against Alzheimer disease.

In another study, peripherally administered antibodies raised againstAβ₁₋₄₂, were able to cross the blood-brain barrier, bind Aβ peptide, andinduce clearance of pre-existing amyloid (Bard, F. et al., NatureMedicine 6:916-19 (2000)). This study utilized either polyclonalantibodies raised against Aβ₁₋₄₂, or monoclonal antibodies raisedagainst synthetic fragments derived from different regions of Aβ. Thusinduction of antibodies can be considered as a potential therapeutictreatment for Alzheimer disease.

It is well established that the administration of purified proteinsalone is usually not sufficient to elicit a strong immune response;isolated antigen generally must be given together with helper substancescalled adjuvants. Within these adjuvants, the administered antigen isprotected against rapid degradation, and the adjuvant provides anextended release of a low level of antigen.

As indicated, one of the key events in Alzheimer's Disease (AD) is thedeposition of amyloid as insoluble fibrous masses (amyloidogenesis)resulting in extracellular neuritic plaques and deposits around thewalls of cerebral blood vessels (for review see Selkoe, D. J. (1999)Nature. 399, A23-31). The major constituent of the neuritic plaques andcongophilic angiopathy is amyloid β (Aβ), although these deposits alsocontain other proteins such as glycosaminoglycans and apolipoproteins.Aβ is proteolytically cleaved from a much larger glycoprotein known asAmyloid Precursor Proteins (APPs), which comprises isoforms of 695-770amino acids with a single hydrophobic transmembrane region. Aβ forms agroup of peptides up to 43 amino acids in length showing considerableamino- and carboxy-terminal heterogeneity (truncation) as well asmodifications (Roher, A. E., Palmer, K. C., Chau, V., & Ball, M. J.(1988) J. Cell Biol. 107, 2703-2716. Roher, A. E., Palmer, K. C.,Yurewicz, E. C., Ball, M. J., & Greenberg, B. D. (1993) J. Neurochem.61, 1916-1926). Prominent isoforms are Aβ1-40 and 1-42. It has a highpropensity to form B-sheets aggregating into fibrils, which ultimatelyleads to the amyloid. Recent studies demonstrated that avaccination-induced reduction in brain amyloid deposits resulted incognitive improvements (Schenk, D., Barbour, R., Dunn, W., Gordon, G.,Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K.,et al. (1999) Nature. 400, 173-177).

We have surprisingly found that self-molecules or self-antigenspresented in a highly ordered and repetitive array were able toefficiently induce self-specific immune responses, in particularantibody responses. Moreover, such responses could even be induced inthe absence of adjuvants that otherwise non-specifically activateantigen presenting cells and other immune cells.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compositions, which comprises highlyordered and repetitive antigen or antigenic determinant arrays, as wellas the processes for their production and their uses. Thus, thecompositions of the invention are useful for the production of vaccinesfor the prevention of infectious diseases, the treatment of allergiesand cancers, and to efficiently induce self-specific immune responses,in particular antibody responses.

In a first aspect, the present invention provides a novel compositioncomprising, or alternatively consisting of, (A) a non-natural molecularscaffold and (B) an antigen or antigenic determinant. The non-naturalmolecular scaffold comprises, or alternatively consists of, (i) a coreparticle selected from the group consisting of (1) a core particle ofnon-natural origin and (2) a core particle of natural origin; and (ii)an organizer comprising at least one first attachment site, wherein saidorganizer is connected to said core particle by at least one covalentbond. The antigen or antigenic determinant is a self antigen or afragment thereof and has at least one second attachment site which isselected from the group consisting of (i) an attachment site notnaturally occurring with said antigen or antigenic determinant; and (ii)an attachment site naturally occurring with said antigen or antigenicdeterminant. The invention provides for an ordered and repetitive selfantigen array through an association of the second attachment site tothe first attachment site by way of at least one non-peptide bond. Thus,the self antigen or self antigenic determinant and the non-naturalmolecular scaffold are brought together through this association of thefirst and the second attachment site to form an ordered and repetitiveantigen array.

In a second aspect, the present invention provides a novel compositioncomprising, or alternatively consisting of, (A) a non-natural molecularscaffold and (B) an antigen or antigenic determinant. The non-naturalmolecular scaffold comprises, or alternatively consists of, (i) a coreparticle and (ii) an organizer comprising at least one first attachmentsite, wherein said core particle is a virus-like particle comprisingrecombinant proteins, or fragments thereof, of a bacteriophage, andwherein said organizer is connected to said core particle by at leastone covalent bond. The antigen or antigenic determinant has at least onesecond attachment site which is selected from the group consisting of(i) an attachment site not naturally occurring with said antigen orantigenic determinant; and (ii) an attachment site naturally occurringwith said antigen or antigenic determinant. The invention provides foran ordered and repetitive antigen array through an association of thesecond attachment site to the first attachment site by way of at leastone non-peptide bond.

In a third aspect, the present invention provides a novel compositioncomprising, or alternatively consisting of, (A) a non-natural molecularscaffold and (B) an antigen or antigenic determinant. The non-naturalmolecular scaffold comprises, or alternatively consists of, (i) a coreparticle selected from the group consisting of (1) a core particle ofnon-natural origin and (2) a core particle of natural origin; and (ii)an organizer comprising at least one first attachment site, wherein saidorganizer is connected to said core particle by at least one covalentbond. The antigen or antigenic determinant is an amyloid beta peptide(Aβ₁₋₄₂) or a fragment thereof, and has at least one second attachmentsite which is selected from the group consisting of (i) an attachmentsite not naturally occurring with said antigen or antigenic determinant;and (ii) an attachment site naturally occurring with said antigen orantigenic determinant. The invention provides for an ordered andrepetitive antigen array through an association of the second attachmentsite to the first attachment site by way of at least one non-peptidebond.

In a fourth aspect, the present invention provides a novel compositioncomprising, or alternatively consisting of, (A) a non-natural molecularscaffold and (B) an antigen or antigenic determinant. The non-naturalmolecular scaffold comprises, or alternatively consists of, (i) a coreparticle selected from the group consisting of (1) a core particle ofnon-natural origin and (2) a core particle of natural origin; and (ii)an organizer comprising at least one first attachment site, wherein saidorganizer is connected to said core particle by at least one covalentbond. The antigen or antigenic determinant is an anti-idiotypic antibodyor an anti-idiotypic antibody fragment and has at least one secondattachment site which is selected from the group consisting of (i) anattachment site not naturally occurring with said antigen or antigenicdeterminant; and (ii) an attachment site naturally occurring with saidantigen or antigenic determinant. The invention provides for an orderedand repetitive antigen array through an association of the secondattachment site to the first attachment site by way of at least onenon-peptide bond.

Further aspects as well as preferred embodiments and advantages of thepresent invention will become apparent in the following as well as, inparticular, in the light of the detailed description, the examples andthe accompanying claims.

In a preferred embodiment of the present invention, the core particle isa virus-like particle comprising recombinant proteins of a RNA-phage,preferably selected from the group consisting of a) bacteriophage Qβ; b)bacteriophage R17; c) bacteriophage fr; d) bacteriophage GA; e)bacteriophage SP; f) bacteriophage MS2; g) bacteriophage M11; h)bacteriophage MX1; i) bacteriophage NL95; k) bacteriophage f2; and l)bacteriophage PP7. Most preferred are bacteriophage Qβ and bacteriophagefr.

In another preferred embodiment of the invention, the recombinantproteins of the RNA-phages comprise wild type coat proteins.

In further preferred embodiment of the invention, the recombinantproteins of the RNA-phages comprise mutant coat proteins.

In yet another embodiment, the core particle comprises, or alternativelyconsists of, one or more different Hepatitis core (capsid) proteins(HBcAgs). In a related embodiment, one or more cysteine residues ofthese HBcAgs are either deleted or substituted with another amino acidresidue (e.g., a serine residue). In a specific embodiment, the cysteineresidues of the HBcAg used to prepare compositions of the inventionwhich correspond to amino acid residues 48 and 107 in SEQ ID NO:134 areeither deleted or substituted with another amino acid residue (e.g., aserine residue).

Further, the HBcAg variants used to prepare compositions of theinvention will generally be variants which retain the ability toassociate with other HBcAgs to form dimeric or multimeric structuresthat present ordered and repetitive antigen or antigenic determinantarrays.

In another embodiment, the non-natural molecular scaffold comprises, oralternatively consists of, pili or pilus-like structures that have beeneither produced from pilin proteins or harvested from bacteria. Whenpili or pilus-like structures are used to prepare compositions of theinvention, they may be formed from products of pilin genes which arenaturally resident in the bacterial cells but have been modified bygenetically engineered (e.g., by homologous recombination) or pilingenes which have been introduced into these cells.

In a related embodiment, the core particle comprises, or alternativelyconsists of, pili or pilus-like structures that have been eitherprepared from pilin proteins or harvested from bacteria. These coreparticles may be formed from products of pilin genes naturally residentin the bacterial cells.

In a particular embodiment, the organizer may comprise at least onefirst attachment site. The first and the second attachment sites areparticularly important elements of compositions of the invention. Invarious embodiments of the invention, the first and/or the secondattachment site may be an antigen and an antibody or antibody fragmentthereto; biotin and avidin; strepavidin and biotin; a receptor and itsligand; a ligand-binding protein and its ligand; interacting leucinezipper polypeptides; an amino group and a chemical group reactivethereto; a carboxyl group and a chemical group reactive thereto; asulfhydryl group and a chemical group reactive thereto; or a combinationthereof.

In a further preferred embodiment, the composition further comprises anamino acid linker. Preferably the amino acid linker comprises, oralternatively consists of, the second attachment site. The secondattachment site mediates a directed and ordered association and binding,respectively, of the antigen to the core particle. An important functionof the amino acid linker is to further ensure proper display andaccessibility of the second attachment site, and thus to facilitate thebinding of the antigen to the core particle, in particular by way ofchemical cross-linking. Another important property of the amino acidlinker is to further ensure optimal accessibility and, in particular,reactivity of the second attachment site. These properties of the aminoacid linker are of even more importance for protein antigens.

In another preferred embodiment, the amino acid linker is selected fromthe group consisting of (a) CGG; (b) N-terminal gamma 1-linker; (c)N-terminal gamma 3-linker; (d) Ig hinge regions; (e) N-terminal glycinelinkers; (f) (G)_(k)C(G)_(n) with n=0-12 and k=0-5; (g) N-terminalglycine-serine linkers; (h) (G)_(k)C(G)_(m)(S)_(l)(GGGGS)_(n) withn=0-3, k=0-5, m=0-10, 1=0-2 (SEQ ID NO:424); (i) GGC; (k) GGC-NH2; (l)C-terminal gamma 1-linker; (m) C-terminal gamma 3-linker; (n) C-terminalglycine linkers; (o) (G)_(n)C(G)_(k) with n=0-12 and k=0-5; (p)C-terminal glycine-serine linkers; (q)(G)_(m)(S)_(l)(GGGGS)_(n)(G)_(o)C(G)_(k) with n=0-3, k=0-5, m=0-10,l=0-2, and o=0-8 (SEQ ID NO:425).

An important property of glycine and glycine serine linkers is theirflexibility, in particular their structural flexibility, allowing a widerange of conformations and disfavoring folding into structuresprecluding accessibility of the second attachment site. As glycine andglycine serine linkers contain either no or a limited amount of sidechain residues, they have limited tendency for engagement into extensiveinteractions with the antigen, thus, further ensuring accessibility ofthe second attachment site. Serine residues within the glycine serinelinkers confer improved solubility properties to these linkers.Accordingly, the insertion of one or two amino acids either in tandem orisolation, and in particular of polar or charged amino acid residues, inthe glycine or glycine serine amino acid linker, is also encompassed bythe teaching of the invention.

In a further preferred embodiment, the amino acid linker is eitherGGC-NH2, GGC-NMe, GGC-N(Me)2, GGC-NHET or GGC-N(Et)2, in which theC-terminus of the cysteine residue of GGC is amidated. These amino acidlinkers are preferred in particular for peptide antigens, and inparticular for embodiments, in which the antigen or antigenicdeterminant with said second attachment site comprises Aβ peptides orfragments thereof. Particular preferred is GGC-NH2. In anotherembodiment, the amino acid linker is an Immunoglobulin (Ig) hingeregion. Fragments of Ig hinge regions are also within the scope of theinvention, as well as Ig hinge regions modified with glycine residues.Preferably, the Ig hinge regions contain only one cysteine residue. Itis to be understood, that the single cysteine residue of the Ig hingeregion amino acid linker can be located at several positions within thelinker sequence, and a man skilled in the art would know how to selectthem with the guidance of the teachings of this invention.

In one embodiment, the invention provides the coupling of almost anyantigen of choice to the surface of a virus, bacterial pilus, structureformed from bacterial pilin, bacteriophage, virus-like particle or viralcapsid particle. By bringing an antigen into a quasi-crystalline‘virus-like’ structure, the invention exploits the strong antiviralimmune reaction of a host for the production of a highly efficientimmune response, i.e., a vaccination, against the displayed antigen.

In yet another embodiment, the antigen may be selected from the groupconsisting of: (1) a protein suited to induce an immune response againstcancer cells; (2) a protein suited to induce an immune response againstinfectious diseases; (3) a protein suited to induce an immune responseagainst allergens; (4) a protein suited to induce an improved responseagainst self-antigens; and (5) a protein suited to induce an immuneresponse in farm animals or pets. In another embodiment, the firstattachment site and/or the second attachment site are selected from thegroup comprising: (1) a genetically engineered lysine residue and (2) agenetically engineered cysteine residue, two residues that may bechemically linked together.

In a yet further preferred embodiment, first attachment site comprisesor is an amino group and said second attachment site comprises or is asulfhydryl group. Preferably, the first attachment site comprises or isa lysine residue and said second attachment site comprises or is acysteine residue.

The invention also includes embodiments where the organizer particle hasonly a single first attachment site and the antigen or antigenicdeterminant has only a single second attachment site. Thus, when anordered and repetitive antigen array is prepared using such embodiments,each organizer will be bound to a single antigen or antigenicdeterminant.

In a further aspect, the invention provides compositions comprising, oralternatively consisting of, (a) a non-natural molecular scaffoldcomprising (i) a core particle selected from the group consisting of acore particle of non-natural origin and a core particle of naturalorigin, and (ii) an organizer comprising at least one first attachmentsite, wherein the core particle comprises, or alternatively consists of,a virus-like particle, a bacterial pilus, a pilus-like structure, or amodified HBcAg, or fragment thereof, and wherein the organizer isconnected to the core particle by at least one covalent bond, and (b) anantigen or antigenic determinant with at least one second attachmentsite, the second attachment site being selected from the groupconsisting of (i) an attachment site not naturally occurring with theantigen or antigenic determinant and (ii) an attachment site naturallyoccurring with the antigen or antigenic determinant, wherein the secondattachment site is capable of association through at least onenon-peptide bond to the first attachment site, and wherein the antigenor antigenic determinant and the scaffold interact through theassociation to form an ordered and repetitive antigen array.

Other embodiments of the invention include processes for the productionof compositions of the invention and a methods of medical treatmentusing vaccine compositions described herein.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide further explanation of the invention asclaimed.

In a still further aspect, the present invention provides a compositioncomprising a bacteriophage Qβ coat protein attached by a covalent bondto phospholipase A₂ protein, or a fragment thereof. In a preferredembodiment, the phospholipase A₂ protein, or a fragment thereof, and thebacteriophage Qβ coat protein interact through the covalent bond to forman ordered and repetitive antigen array. In another preferredembodiment, the covalent bond is not a peptide bond. In anotherpreferred embodiment, the phospholipase A₂ protein includes an aminoacid selected from the group consisting of the amino acid sequence ofSEQ ID NO:168, the amino acid sequence of SEQ ID NO:169, the amino acidsequence of SEQ ID NO:170, the amino acid sequence of SEQ ID NO:171, theamino acid sequence of SEQ ID NO:172, the amino acid sequence of SEQ IDNO:173, the amino acid sequence of SEQ ID NO:174, and the amino acidsequence of SEQ ID NO:175.

The present invention also provides a method of making the compositioncomprising combining the bacteriophage Qβ coat protein and thephospholipase A₂ protein, wherein the bacteriophage Qβ coat protein andthe phospholipase A₂ protein interact to form an antigen array.

In another aspect, the present invention also provides a compositioncomprising a non-natural molecular scaffold comprising a bacteriophageQβ coat protein, and an organizer comprising at least one firstattachment site, wherein the organizer is connected to the bacteriophageQβ coat protein by at least one covalent bond; and phospholipase A₂protein, or a fragment thereof, or a variant thereof with at least onesecond attachment site, the second attachment site being selected fromthe group consisting of: an attachment site not naturally occurring withthe a phospholipase A₂ protein, or a fragment thereof; and an attachmentsite naturally occurring with the a phospholipase A₂ protein, or afragment thereof, wherein the second attachment site associates throughat least one non-peptide bond to the first attachment site, and whereinthe antigen or antigenic determinant and the scaffold interact throughthe association to form an ordered and repetitive antigen array. In apreferred embodiment, the phospholipase A₂ protein includes an aminoacid selected from the group consisting of the amino acid sequence ofSEQ ID NO:168, the amino acid sequence of SEQ ID NO:169, the amino acidsequence of SEQ ID NO:170, the amino acid sequence of SEQ ID NO:171, theamino acid sequence of SEQ ID NO:172, the amino acid sequence of SEQ IDNO:173, the amino acid sequence of SEQ ID NO:174, and the amino acidsequence of SEQ ID NO:175.

The present invention also provides a method of making the compositioncomprising combining the bacteriophage Qβ coat protein and thephospholipase A₂ protein, wherein the bacteriophage Qβ coat protein andthe phospholipase A₂ protein interact to form an antigen array.Preferably, the antigen array is ordered and/or repetitive.

The present invention also provides a pharmaceutical compositioncomprising a phospholipase A₂ protein, and a pharmaceutically acceptablecarrier. The present invention also provides a vaccine compositioncomprising a phospholipase A₂ protein. In a preferred embodiment, thevaccine composition of claim 31, further comprising at least oneadjuvant.

The present invention also provides a method of treating an allergy tobee venom, comprising administering the pharmaceutical composition orthe vaccine composition to a subject. As a result of such administrationthe subject exhibits a decreased immune response to the venom.

The invention also relates to a vaccine for the prevention ofprion-mediated diseases by induction of anti-lymphotoxinβ,anti-lymphotoxinα or anti-lymphotoxinβ-receptor antibodies. The vaccinecontains protein carries foreign to the immunized human or animalcoupled to lymphotoxinβ or fragments thereof, lymphotoxinα or fragmentsthereof or the lymphotoxinβ receptor or fragments thereof. The vaccineis injected in humans or animals in order to induce antibodies specificfor endogenous lymphotoxinβ, lymphotoxinα or lymphotoxinβ receptor.These induced anti-lymphotoxinβ, lymphotoxinα or anti-lymphotoxinβreceptor antibodies reduce or eliminate the pool of follicular dendriticcells present in lymphoid organs. Since prion-replication in lymphoidorgans and transport to the central nervous system are impaired in theabsence of follicular dendritic cells, this treatment inhibitsprogression of prion-mediated disease. In addition, blockinglymphotoxinβ is beneficial for patients with autoimmune diseases such asdiabetes type I.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1C Modular eukaryotic expression vectors for expression ofantigens according to the invention (FIG. 1A: SEQ ID NO:426; FIG. 1B:SEQ ID NO:427; FIG. 1C: SEQ ID NO:428);

FIG. 2A-2C Cloning, expression and coupling of resistin to Qβ capsidprotein FIG. 2A: SEQ ID NO:429; FIG. 2B: SEQ ID NO:430);

FIG. 3A-3B Cloning and expression of lymphotoxin-β constructs forcoupling to virus-like particles and pili.

FIG. 4A-4B Cloning, expression and coupling of MIF constructs to Qβcapsid protein.

FIG. 4C ELISA analysis of IgG antibodies specific for MIF in sera ofmice immunized against MIF proteins coupled to Qβ capsid protein.

FIG. 5 Coupling of MIF constructs to fr capsid protein and toHBcAg-lys-2cys-Mut capsid protein analyzed by SDS-Page.

FIG. 6 Cloning and expression of human-C-RANKL.

FIG. 7 Cloning and expression of prion protein.

FIG. 8A. ELISA analysis of IgG antibodies specific for “Angio I” in seraof mice immunized against angiotensin peptides coupled to Qβ capsidprotein.

FIG. 8B. ELISA analysis of IgG antibodies specific for “Angio II” insera of mice immunized against angiotensin peptides coupled to Qβ capsidprotein.

FIG. 8C. ELISA analysis of IgG antibodies specific for “Angio III” insera of mice immunized against angiotensin peptides coupled to Qβ capsidprotein.

FIG. 8D. ELISA analysis of IgG antibodies specific for “Angio IV” insera of mice immunized against angiotensin peptides coupled to Qβ capsidprotein.

FIG. 9A. ELISA analysis of IgG antibodies specific for “Der p I p52” insera of mice immunized against Der p I peptides coupled to Qβ capsidprotein.

FIG. 9B. ELISA analysis of IgG antibodies specific for “Der p I p117” insera of mice immunized against Der p I peptides coupled to Qβ capsidprotein.

FIG. 10A. ELISA analysis of IgG antibodies specific for human VEGFR IIpeptide in sera of mice immunized against human VEGFR II peptide and theextracellular domain of human VEGFR II both coupled to Type-1 piliprotein.

FIG. 10B. ELISA analysis of IgG antibodies specific for theextracellular domain of human VEGFR II in sera of mice immunized againsthuman VEGFR II peptide and extracellular domain of human VEGFR II bothcoupled to Type-1 pili protein.

FIG. 11. ELISA analysis of IgG antibodies specific for anti-TNFα proteinin sera of mice immunized against full length HBc-TNF.

FIG. 12. ELISA analysis of IgG antibodies specific for anti-TNFα proteinin sera of mice immunized against 2cysLys-mut HBcAg1-149 coupled to the3′TNF II peptide

FIG. 13A. SDS-PAGE analysis of coupling of “Aβ1-15” to Qβ capsid proteinusing the cross-linker SMPH.

FIG. 13B. SDS-PAGE analysis of coupling of “Aβ33-42” to Qβ capsidprotein using the cross-linker SMPH.

FIG. 13C. SDS-PAGE analysis of coupling of “Aβ1-27” to Qβ capsid proteinusing the cross-linker SMPH.

FIG. 13D. SDS-PAGE analysis of coupling of “Aβ1-15” to Qβ capsid proteinusing the cross-linker Sulfo-GMBS.

FIG. 13E. SDS-PAGE analysis of coupling of “Aβ1-15” to Qβ capsid proteinusing the cross-linker Sulfo-MBS.

FIG. 14A. ELISA analysis of IgG antibodies specific for “Aβ1-15” in seraof mice immunized against “Aβ1-15” coupled to Qβ capsid protein.

FIG. 14B. ELISA analysis of IgG antibodies specific for “Aβ1-27” in seraof mice immunized against “Aβ1-27” coupled to Qβ capsid protein.

FIG. 14C. ELISA analysis of IgG antibodies specific for “Aβ33-42” insera of mice immunized against “Aβ33-42” coupled to Qβ capsid protein.

FIG. 15A. SDS-PAGE analysis of coupling of pCC2 to Qβ capsid protein.

FIG. 15B. SDS-PAGE analysis of coupling of pCA2 to Qβ capsid protein.

FIG. 15C. SDS-PAGE analysis of coupling of pCB2 to Qβ capsid protein.

FIGS. 16A and 16B Coupling of prion peptides to Qβ capsid protein;SDS-Page analysis.

FIG. 17 A. SDS-PAGE analysis of expression of IL-5 in bacteria

FIG. 17 B. Western-Blot analysis of expression of IL-5 and IL-13 ineukaryotic cells

FIG. 18 A. SDS-PAGE analysis of coupling of murine VEGFR-2 peptide toPili.

FIG. 18 B. SDS-PAGE analysis of coupling of murine VEGFR-2 peptide to Qβcapsid protein.

FIG. 18 C. SDS-PAGE analysis of coupling of murine VEGFR-2 peptide toHBcAg-lys-2cys-Mut.

FIG. 18 D. ELISA analysis of IgG antibodies specific for murine VEGFR-2peptide in sera of mice immunized against murine VEGFR-2 peptide coupledto Pili.

FIG. 18 E. ELISA analysis of IgG antibodies specific for murine VEGFR-2peptide in sera of mice immunized against murine VEGFR-2 peptide coupledto Qβ capsid protein.

FIG. 18 F. ELISA analysis of IgG antibodies specific for murine VEGFR-2peptide in sera of mice immunized against murine VEGFR-2 peptide coupledto HBcAg-lys-2cys-Mut.

FIG. 19 A. SDS-PAGE analysis of coupling of Aβ 1-15 peptide toHBcAg-lys-2cys-Mut and fr capsid protein.

FIG. 19 B. ELISA analysis of IgG antibodies specific for Aβ 1-15 peptidein sera of mice immunized against Aβ 1-15 peptide coupled toHBcAg-lys-2cys-Mut or fr capsid protein.

FIG. 20 ELISA analysis of IgG antibodies specific for human Aβ in seraof transgenic APP23 mice immunized with human Aβ peptides coupled to Qβcapsid protein.

FIG. 21 SDS-PAGE analysis of coupling of an Fab antibody fragment to Qβcapsid protein.

FIG. 22 A. SDS-PAGE analysis of coupling of flag peptide coupled tomutant Qβ capsid protein with cross-linker sulfo GMBS

FIG. 22 B. SDS-PAGE analysis of coupling of flag peptide coupled tomutant Qβ capsid protein with cross-linker sulfo MBS

FIG. 22 C. SDS-PAGE analysis of coupling of flag peptide coupled tomutant Qβ capsid protein with cross-linker SMPH

FIG. 22 D. SDS-PAGE analysis of coupling of PLA₂-cys protein coupled tomutant Qβ capsid protein with cross-linker SMPH

FIG. 23 ELISA analysis of immunization with M2 peptide coupled to mutantQβ capsid protein and fr capsid

FIG. 24 SDS-PAGE analysis of coupling of DER p1,2 peptide coupled tomutant Qβ capsid protein

FIG. 25 A Desensitization of allergic mice with PLA2 coupled to Qβcapsid protein: temperature measurements

FIG. 25 B Desensitization of allergic mice with PLA2-cys coupled to Qβcapsid protein: IgG 2A and Ig E titers

FIG. 26 SDS-PAGE Analysis and Western-blot analysis of coupling ofPLA₂-cys to Qβ capsid protein

FIG. 27 A ELISA analysis of IgG antibodies specific for M2 peptide insera of mice immunized against M2 peptide coupled to HBcAg-lys-2cys-Mut,Qβ capsid protein, fr capsid protein, HBcAg-lys-1-183 and M2 peptidefused to HBcAg 1-183

FIG. 27B Survival of mice vaccinated intravenously followed by a lethalinfluenza challenge.

FIG. 28 A SDS-PAGE Analysis of coupling of anti-idiotypic IgE mimobodyVAE051 to Qβ capsid protein

FIG. 28 B. ELISA analysis of IgG antibodies specific for anti-idiotypicantibody VAE051 and Human IgE in sera of mice immunized against VAE051coupled to Qβ capsid protein

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

Alphavirus: As used herein, the term “alphavirus” refers to any of theRNA viruses included within the genus Alphavirus. Descriptions of themembers of this genus are contained in Strauss and Strauss, Microbiol.Rev., 58:491-562 (1994). Examples of alphaviruses include Aura virus,Bebaru virus, Cabassou virus, Chikungunya virus, Easter equineencephalomyelitis virus, Fort morgan virus, Getah virus, Kyzylagachvirus, Mayoaro virus, Middleburg virus, Mucambo virus, Ndumu virus,Pixuna virus, Tonate virus, Triniti virus, Una virus, Western equineencephalomyelitis virus, Whataroa virus, Sindbis virus (SIN), Semlikiforest virus (SFV), Venezuelan equine encephalomyelitis virus (VEE), andRoss River virus.

Antigen: As used herein, the term “antigen” is a molecule capable ofbeing bound by an antibody. An antigen is additionally capable ofinducing a humoral immune response and/or cellular immune responseleading to the production of B- and/or T-lymphocytes. An antigen mayhave one or more epitopes (B- and T-epitopes). The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

Antigenic determinant: As used herein, the term“antigenic determinant”is meant to refer to that portion of an antigen that is specificallyrecognized by either B- or T-lymphocytes. B-lymphocytes respond toforeign antigenic determinants via antibody production, whereasT-lymphocytes are the mediator of cellular immunity. Thus, antigenicdeterminants or epitopes are those parts of an antigen that arerecognized by antibodies, or in the context of an MHC, by T-cellreceptors.

Association: As used herein, the term “association” as it applies to thefirst and second attachment sites, refers to at least one non-peptidebond. The nature of the association may be covalent, ionic, hydrophobic,polar or any combination thereof

Attachment Site, First: As used herein, the phrase “first attachmentsite” refers to an element of the “organizer”, itself bound to the coreparticle in a non-random fashion, to which the second attachment sitelocated on the antigen or antigenic determinant may associate. The firstattachment site may be a protein, a polypeptide, an amino acid, apeptide, a sugar, a polynucleotide, a natural or synthetic polymer, asecondary metabolite or compound (biotin, fluorescein, retinol,digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a combinationthereof, or a chemically reactive group thereof. Multiple firstattachment sites are present on the surface of the non-natural molecularscaffold in a repetitive configuration.

Attachment Site, Second: As used herein, the phrase “second attachmentsite” refers to an element associated with the antigen or antigenicdeterminant to which the first attachment site of the “organizer”located on the surface of the non-natural molecular scaffold mayassociate. The second attachment site of the antigen or antigenicdeterminant may be a protein, a polypeptide, a peptide, a sugar, apolynucleotide, a natural or synthetic polymer, a secondary metaboliteor compound (biotin, fluorescein, retinol, digoxigenin, metal ions,phenylmethylsulfonylfluoride), or a combination thereof, or a chemicallyreactive group thereof. At least one second attachment site is presenton the antigen or antigenic determinant. The term “antigen or antigenicdeterminant with at least one second attachment site” refers, therefore,to an antigen or antigenic construct comprising at least the antigen orantigenic determinant and the second attachment site. However, inparticular for a second attachment site, which is not naturallyoccurring within the antigen or antigenic determinant, these antigen orantigenic constructs comprise an “amino acid linker”. Such an amino acidlinker, or also just termed “linker” within this specification, eitherassociates the antigen or antigenic determinant with the secondattachment site, or more preferably, already comprises or contains thesecond attachment site, typically—but not necessarily—as one amino acidresidue, preferably as a cysteine residue. The term “amino acid linker”as used herein, however, does not intend to imply that such an aminoacid linker consists exclusively of amino acid residues, even if anamino acid linker consisting of amino acid residues is a preferredembodiment of the present invention. The amino acid residues of theamino acid linker is, preferably, composed of naturally occurring aminoacids or unnatural amino acids known in the art, all-L or all-D ormixtures thereof. However, an amino acid linker comprising a moleculewith a sulfhydryl group or cysteine residue is also encompassed withinthe invention. Such a molecule comprise preferably a C1-C6 alkyl-,cycloalkyl (C5,C6), aryl or heteroaryl moiety. Association between theantigen or antigenic determinant or optionally the second attachmentsite and the amino acid linker is preferably by way of at least onecovalent bond, more preferably by way of at least one peptide bond.

Bound: As used herein, the term “bound” refers to binding or attachmentthat may be covalent, e.g., by chemically coupling, or non-covalent,e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc.Covalent bonds can be, for example, ester, ether, phosphoester, amide,peptide, imide, carbon-sulfur bonds, carbon-phosphorus bonds, and thelike. The term “bound” is broader than and includes terms such as“coupled,” “fused” and “attached”.

Core particle: As used herein, the term “core particle” refers to arigid structure with an inherent repetitive organization that provides afoundation for attachment of an “organizer”. A core particle as usedherein may be the product of a synthetic process or the product of abiological process.

Coat protein(s): As used herein, the term “coat protein(s)” refers tothe protein(s) of a bacteriophage or a RNA-phage capable of beingincorporated within the capsid assembly of the bacteriophage or theRNA-phage. However, when referring to the specific gene product of thecoat protein gene of RNA-phages the term “CP” is used. For example, thespecific gene product of the coat protein gene of RNA-phage Qβ isreferred to as “Qβ CP”, whereas the “coat proteins” of bacteriophage Qbcomprise the “Qβ CP” as well as the A1 protein.

Cis-acting: As used herein, the phrase “cis-acting” sequence refers tonucleic acid sequences to which a replicase binds to catalyze theRNA-dependent replication of RNA molecules. These replication eventsresult in the replication of the full-length and partial RNA moleculesand, thus, the alpahvirus subgenomic promoter is also a “cis-acting”sequence. Cis-acting sequences may be located at or near the 5′ end, 3′end, or both ends of a nucleic acid molecule, as well as internally.

Fusion: As used herein, the term “fusion” refers to the combination ofamino acid sequences of different origin in one polypeptide chain byin-frame combination of their coding nucleotide sequences. The term“fusion” explicitly encompasses internal fusions, i.e., insertion ofsequences of different origin within a polypeptide chain, in addition tofusion to one of its termini.

Heterologous sequence: As used herein, the term “heterologous sequence”refers to a second nucleotide sequence present in a vector of theinvention. The term “heterologous sequence” also refers to any aminoacid or RNA sequence encoded by a heterologous DNA sequence contained ina vector of the invention. Heterologous nucleotide sequences can encodeproteins or RNA molecules normally expressed in the cell type in whichthey are present or molecules not normally expressed therein (e.g.,Sindbis structural proteins).

Isolated: As used herein, when the term “isolated” is used in referenceto a molecule, the term means that the molecule has been removed fromits native environment. For example, a polynucleotide or a polypeptidenaturally present in a living animal is not “isolated,” but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated.” Further, recombinant DNA moleculescontained in a vector are considered isolated for the purposes of thepresent invention. Isolated RNA molecules include in vivo or in vitroRNA replication products of DNA and RNA molecules. Isolated nucleic acidmolecules further include synthetically produced molecules.Additionally, vector molecules contained in recombinant host cells arealso isolated. Thus, not all “isolated” molecules need be “purified.”

Immunotherapeutic: As used herein, the term “immunotherapeutic” is acomposition for the treatment of diseases or disorders. Morespecifically, the term is used to refer to a method of treatment forallergies or a method of treatment for cancer.

Individual: As used herein, the term “individual” refers tomulticellular organisms and includes both plants and animals. Preferredmulticellular organisms are animals, more preferred are vertebrates,even more preferred are mammals, and most preferred are humans.

Low or undetectable: As used herein, the phrase “low or undetectable,”when used in reference to gene expression level, refers to a level ofexpression which is either significantly lower than that seen when thegene is maximally induced (e.g., at least five fold lower) or is notreadily detectable by the methods used in the following examplessection.

Lectin: As used herein, proteins obtained particularly from the seeds ofleguminous plants, but also from many other plant and animal sources,that have binding sites for specific mono- or oligosaccharides. Examplesinclude concanavalin A and wheat-germ agglutinin, which are widely usedas analytical and preparative agents in the study of glycoprotein.

Mimotope: As used herein, the term“mimotope” refers to a substance whichinduces an immune response to an antigen or antigenic determinant.Generally, the term mimotope will be used with reference to a particularantigen. For example, a peptide which elicits the production ofantibodies to a phospholipase A₂ (PLA₂) is a mimotope of the antigenicdeterminant to which the antibodies bind. A mimotope may or may not havesubstantial structural similarity to or share structural properties withan antigen or antigenic determinant to which it induces an immuneresponse. Methods for generating and identifying mimotopes which induceimmune responses to particular antigens or antigenic determinants areknown in the art and are described elsewhere herein.

Natural origin: As used herein, the term “natural origin” means that thewhole or parts thereof are not synthetic and exist or are produced innature.

Non-natural: As used herein, the term generally means not from nature,more specifically, the term means from the hand of man.

Non-natural origin: As used herein, the term “non-natural origin”generally means synthetic or not from nature; more specifically, theterm means from the hand of man.

Non-natural molecular scaffold: As used herein, the phrase “non-naturalmolecular scaffold” refers to any product made by the hand of man thatmay serve to provide a rigid and repetitive array of first attachmentsites. Ideally but not necessarily, these first attachment sites are ina geometric order. The non-natural molecular scaffold may be organic ornon-organic and may be synthesized chemically or through a biologicalprocess, in part or in whole. The non-natural molecular scaffold iscomprised of: (a) a core particle, either of natural or non-naturalorigin; and (b) an organizer, which itself comprises at least one firstattachment site and is connected to a core particle by at least onecovalent bond. In a particular embodiment, the non-natural molecularscaffold may be a virus, virus-like particle, a bacterial pilus, a viruscapsid particle, a phage, a recombinant form thereof, or syntheticparticle.

Ordered and repetitive antigen or antigenic determinant array: As usedherein, the term “ordered and repetitive antigen or antigenicdeterminant array” generally refers to a repeating pattern of antigen orantigenic determinant, characterized by a uniform spacial arrangement ofthe antigens or antigenic determinants with respect to the non-naturalmolecular scaffold. In one embodiment of the invention, the repeatingpattern may be a geometric pattern. Examples of suitable ordered andrepetitive antigen or antigenic determinant arrays are those whichpossess strictly repetitive paracrystalline orders of antigens orantigenic determinants with spacings of 5 to 15 nanometers.

Organizer: As used herein, the term “organizer” is used to refer to anelement bound to a core particle in a non-random fashion that provides anucleation site for creating an ordered and repetitive antigen array. Anorganizer is any element comprising at least one first attachment sitethat is bound to a core particle by at least one covalent bond. Anorganizer may be a protein, a polypeptide, a peptide, an amino acid(i.e., a residue of a protein, a polypeptide or peptide), a sugar, apolynucleotide, a natural or synthetic polymer, a secondary metaboliteor compound (biotin, fluorescein, retinol, digoxigenin, metal ions,phenylmethylsulfonylfluoride), or a combination thereof, or a chemicallyreactive group thereof. Therefore, the organizer further ensuresformation of an ordered and repetitive antigen array in accordance withthe present invention. In typical embodiments of the invention, the coreparticle is modified, e.g. by way of genetic engineering or chemicalreaction, so as to generate a non-natural molecular scaffold comprisingthe core particle and the organizer, the latter being connected to thecore particle by at least one covalent bond. In certain embodiments ofthe invention, however, the organizer is selected as being part of thecore particle. Therefore, for those embodiments modification of the coreparticle is not necessarily needed to generate a non-natural molecularscaffold comprising the core particle and the organizer and to ensurethe formation of an ordered and repetitive antigen array.

Permissive temperature: As used herein, the phrase “permissivetemperature” refers to temperatures at which an enzyme has relativelyhigh levels of catalytic activity.

Pili: As used herein, the term “pili” (singular being “pilus”) refers toextracellular structures of bacterial cells composed of protein monomers(e.g., pilin monomers) which are organized into ordered and repetitivepatterns. Further, pili are structures which are involved in processessuch as the attachment of bacterial cells to host cell surfacereceptors, inter-cellular genetic exchanges, and cell-cell recognition.Examples of pili include Type-1 pili, P-pili, F1C pili, S-pili, and987P-pili. Additional examples of pili are set out below.

Pilus-like structure: As used herein, the phrase “pilus-like structure”refers to structures having characteristics similar to that of pili andcomposed of protein monomers. One example of a “pilus-like structure” isa structure formed by a bacterial cell which expresses modified pilinproteins that do not form ordered and repetitive arrays that areessentially identical to those of natural pili.

Polypeptide: As used herein the term “polypeptide” refers to a polymercomposed of amino acid residues, generally natural amino acid residues,linked together through peptide bonds. Although a polypeptide may notnecessarily be limited in size, the term polypeptide is often used inconjunction with peptide of a size of about ten to about 50 amino acids.

Protein: As used herein, the term protein refers to a polypeptidegenerally of a size of above 20, more particularly of above 50 aminoacid residues. Proteins generally have a defined three dimensionalstructure although they do not necessarily need to, and are oftenreferred to as folded, in opposition to peptides and polypeptides whichoften do not possess a defined three-dimensional structure, but rathercan adopt a large number of different conformations, and are referred toas unfolded. The defined three-dimensional structures of proteins isespecially important for the association between the core particle andthe antigen, mediated by the second attachment site, and in particularby way of chemical cross-linking between the first and second attachmentsite using a chemical cross-linker. The amino acid linker is alsointimately related to the structural properties of proteins in someaspects of the invention.

Purified: As used herein, when the term “purified” is used in referenceto a molecule, it means that the concentration of the molecule beingpurified has been increased relative to molecules associated with it inits natural environment. Naturally associated molecules includeproteins, nucleic acids, lipids and sugars but generally do not includewater, buffers, and reagents added to maintain the integrity orfacilitate the purification of the molecule being purified. For example,even if mRNA is diluted with an aqueous solvent during oligo dT columnchromatography, mRNA molecules are purified by this chromatography ifnaturally associated nucleic acids and other biological molecules do notbind to the column and are separated from the subject mRNA molecules.

Receptor: As used herein, the term “receptor” refers to proteins orglycoproteins or fragments thereof capable of interacting with anothermolecule, called the ligand. The ligand may belong to any class ofbiochemical or chemical compounds. The receptor need not necessarily bea membrane-bound protein. Soluble protein, like e.g., maltose bindingprotein or retinol binding protein are receptors as well.

Residue: As used herein, the term “residue” is meant to mean a specificamino acid in a polypeptide backbone or side chain.

Recombinant host cell: As used herein, the term “recombinant host cell”refers to a host cell into which one ore more nucleic acid molecules ofthe invention have been introduced.

Recombinant virus: As used herein, the phrase “recombinant virus” refersto a virus that is genetically modified by the hand of man. The phrasecovers any virus known in the art. More specifically, the phrase refersto a an alphavirus genetically modified by the hand of man, and mostspecifically, the phrase refers to a Sinbis virus genetically modifiedby the hand of man.

Restrictive temperature: As used herein, the phrase “restrictivetemperature” refers to temperatures at which an enzyme has low orundetectable levels of catalytic activity. Both “hot” and “cold”sensitive mutants are known and, thus, a restrictive temperature may behigher or lower than a permissive temperature.

RNA-dependent RNA replication event: As used herein, the phrase“RNA-dependent RNA replication event” refers to processes which resultin the formation of an RNA molecule using an RNA molecule as a template.

RNA-Dependent RNA polymerase: As used herein, the phrase “RNA-DependentRNA polymerase” refers to a polymerase which catalyzes the production ofan RNA molecule from another RNA molecule. This term is used hereinsynonymously with the term “replicase.”

RNA-phage: As used herein, the term “RNA-phage” refers to RNA virusesinfecting bacteria, preferably to single-stranded positive-sense RNAviruses infecting bacteria.

Self antigen: As used herein, the tem “self antigen” refers to proteinsencoded by the host's DNA and products generated by proteins or RNAencoded by the host's DNA are defined as self. In addition, proteinsthat result from a combination of two or several self-molecules or thatrepresent a fraction of a self-molecule and proteins that have a highhomology two self-molecules as defined above (>95%) may also beconsidered self.

Temperature-sensitive: As used herein, the phrase“temperature-sensitive” refers to an enzyme which readily catalyzes areaction at one temperature but catalyzes the same reaction slowly ornot at all at another temperature. An example of a temperature-sensitiveenzyme is the replicase protein encoded by the pCYTts vector, which hasreadily detectable replicase activity at temperatures below 34

C and has low or undetectable activity at 37′ C.

Transcription: As used herein, the term “transcription” refers to theproduction of RNA molecules from DNA templates catalyzed by RNApolymerase.

Untranslated RNA: As used herein, the phrase “untranslated RNA” refersto an RNA sequence or molecule which does not encode an open readingframe or encodes an open reading frame, or portion thereof, but in aformat in which an amino acid sequence will not be produced (e.g., noinitiation codon is present). Examples of such molecules are tRNAmolecules, rRNA molecules, and ribozymes.

Vector: As used herein, the term “vector” refers to an agent (e.g., aplasmid or virus) used to transmit genetic material to a host cell. Avector may be composed of either DNA or RNA.

Virus-like particle: As used herein, the term “virus-like particle”refers to a structure resembling a virus particle. Moreover, avirus-like particle in accordance with the invention is non replicativeand noninfectious since it lacks all or part of the viral genome, inparticular the replicative and infectious components of the viralgenome. A virus-like particle in accordance with the invention maycontain nucleic acid distinct from their genome.

Virus-like particle of a bacteriophage: As used herein, the term“virus-like particle of a bacteriophage” refers to a virus-like particleresembling the structure of a bacteriophage, being non replicative andnoninfectious, and lacking at least the gene or genes encoding for thereplication machinery of the bacteriophage, and typically also lackingthe gene or genes encoding the protein or proteins responsible for viralattachment to or entry into the host. This definition should, however,also encompass virus-like particles of bacteriophages, in which theaforementioned gene or genes are still present but inactive, and,therefore, also leading to non-replicative and noninfectious virus-likeparticles of a bacteriophage.

Virus particle: The term “virus particle” as used herein refers to themorphological form of a virus. In some virus types it comprises a genomesurrounded by a protein capsid; others have additional structures (e.g.,envelopes, tails, etc.).

one, a, or an: When the terms “one,” “a,” or “an” are used in thisdisclosure, they mean “at least one” or “one or more,” unless otherwiseindicated.

2. Compositions of Ordered and Repetitive Antigen or AntigenicDeterminant Arrays and Methods to Make the Same

The disclosed invention provides compositions comprising an ordered andrepetitive antigen or antigenic determinant array. Furthermore, theinvention conveniently enables the practitioner to construct ordered andrepetitive antigen or antigenic determinant arrays for various treatmentpurposes, which includes the prevention of infectious diseases, thetreatment of allergies and the treatment of cancers.

Compositions of the invention essentially comprise, or alternativelyconsist of, two elements: (1) a non-natural molecular scaffold; and (2)an antigen or antigenic determinant with at least one second attachmentsite capable of association through at least one non-peptide bond tosaid first attachment site.

Compositions of the invention also comprise, or alternatively consistof, bacterial pilus proteins to which antigens or antigenic determinantsare directly linked.

The non-natural molecular scaffold comprises, or alternatively consistsof: (a) a core particle selected from the group consisting of (1) a coreparticle of non-natural origin and (2) a core particle of naturalorigin; and (b) an organizer comprising at least one first attachmentsite, wherein said organizer is connected to said core particle by atleast one covalent bond.

Compositions of the invention also comprise, or alternatively consistof, core particles to which antigens or antigenic determinants aredirectly linked.

The antigen or antigenic determinant has at least one second attachmentsite which is selected from the group consisting of (a) an attachmentsite not naturally occurring with said antigen or antigenic determinant;and (b) an attachment site naturally occurring with said antigen orantigenic determinant

The invention provides for an ordered and repetitive antigen arraythrough an association of the second attachment site to the firstattachment site by way of at least one non-peptide bond. Thus, theantigen or antigenic determinant and the non-natural molecular scaffoldare brought together through this association of the first and thesecond attachment site to form an ordered and repetitive antigen array.

The practitioner may specifically design the antigen or antigenicdeterminant and the second attachment site such that the arrangement ofall the antigens or antigenic determinants bound to the non-naturalmolecular scaffold or, in certain embodiments, the core particle will beuniform. For example, one may place a single second attachment site onthe antigen or antigenic determinant at the carboxyl or amino terminus,thereby ensuring through design that all antigen or antigenicdeterminant molecules that are attached to the non-natural molecularscaffold are positioned in a uniform way. Thus, the invention provides aconvenient means of placing any antigen or antigenic determinant onto anon-natural molecular scaffold in a defined order and in a manner whichforms a repetitive pattern.

As will be clear to those skilled in the art, certain embodiments of theinvention involve the use of recombinant nucleic acid technologies suchas cloning, polymerase chain reaction, the purification of DNA and RNA,the expression of recombinant proteins in prokaryotic and eukaryoticcells, etc. Such methodologies are well known to those skilled in theart and may be conveniently found in published laboratory methodsmanuals (e.g., Sambrook, J. et al., eds., MOLECULAR CLONING, ALABORATORY MANUAL, 2nd. edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al., eds., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997)).Fundamental laboratory techniques for working with tissue culture celllines (Celis, J., ed., CELL BIOLOGY, Academic Press, 2^(nd) edition,(1998)) and antibody-based technologies (Harlow, E. and Lane, D.,“Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1988); Deutscher, M. P., “Guide to ProteinPurification,” Meth. Enzymol. 128, Academic Press San Diego (1990);Scopes, R. K., “Protein Purification Principles and Practice,” 3^(rd)ed., Springer-Verlag, New York (1994)) are also adequately described inthe literature, all of which are incorporated herein by reference.

A. Core Particles and Non-Natural Molecular Scaffolds

One element in certain compositions of the invention is a non-naturalmolecular scaffold comprising, or alternatively consisting of, a coreparticle and an organizer. As used herein, the phrase “non-naturalmolecular scaffold” refers to any product made by the hand of man thatmay serve to provide a rigid and repetitive array of first attachmentsites. More specifically, the non-natural molecular scaffold comprises,or alternatively consists of, (a) a core particle selected from thegroup consisting of (1) a core particle of non-natural origin and (2) acore particle of natural origin; and (b) an organizer comprising atleast one first attachment site, wherein said organizer is connected tosaid core particle by at least one covalent bond.

As will be readily apparent to those skilled in the art, the coreparticle of the non-natural molecular scaffold of the invention is notlimited to any specific form. The core particle may be organic ornon-organic and may be synthesized chemically or through a biologicalprocess.

In one embodiment, a non-natural core particle may be a syntheticpolymer, a lipid micelle or a metal. Such core particles are known inthe art, providing a basis from which to build the novel non-naturalmolecular scaffold of the invention. By way of example, syntheticpolymer or metal core particles are described in U.S. Pat. No.5,770,380, which discloses the use of a calixarene organic scaffold towhich is attached a plurality of peptide loops in the creation of an‘antibody mimic’, and U.S. Pat. No. 5,334,394 describes nanocrystallineparticles used as a viral decoy that are composed of a wide variety ofinorganic materials, including metals or ceramics. Suitable metalsinclude chromium, rubidium, iron, zinc, selenium, nickel, gold, silver,platinum. Suitable ceramic materials in this embodiment include silicondioxide, titanium dioxide, aluminum oxide, ruthenium oxide and tinoxide. The core particles of this embodiment may be made from organicmaterials including carbon (diamond). Suitable polymers includepolystyrene, nylon and nitrocellulose. For this type of nanocrystallineparticle, particles made from tin oxide, titanium dioxide or carbon(diamond) are may also be used. A lipid micelle may be prepared by anymeans known in the art. For example micelles may be prepared accordingto the procedure of Baiselle and Millar (Biophys. Chem. 4:355-361(1975)) or Corti et al. (Chem. Phys. Lipids 38:197-214 (1981)) or Lopezet al. (FEBS Lett. 426:314-318 (1998)) or Topchieva and Karezin (J.Colloid Interface Sci. 213:29-35 (1999)) or Morein et al., (Nature308:457-460 (1984)), which are all incorporated herein by reference.

The core particle may also be produced through a biological process,which may be natural or non-natural. By way of example, this type ofembodiment may includes a core particle comprising, or alternativelyconsisting of, a virus, virus-like particle, a bacterial pilus, a phage,a viral capsid particle or a recombinant form thereof. In a morespecific embodiment, the core particle may comprise, or alternativelyconsist of, recombinant proteins of Rotavirus, recombinant proteins ofNorwalk virus, recombinant proteins of Alphavirus, recombinant proteinswhich form bacterial pili or pilus-like structures, recombinant proteinsof Foot and Mouth Disease virus, recombinant proteins of Retrovirus,recombinant proteins of Hepatitis B virus (e.g., a HBcAg), recombinantproteins of Tobacco mosaic virus, recombinant proteins of Flock HouseVirus, and recombinant proteins of human Papillomavirus. The coreparticle may further comprise, or alternatively consist of, one or morefragments of such proteins, as well as variants of such proteins whichretain the ability to associate with each other to form ordered andrepetitive antigen or antigenic determinant arrays.

As explained in more below, variants of proteins which retain theability to associate with each other to form ordered and repetitiveantigen or antigenic determinant arrays may share, for example, at least80%, 85%, 90%, 95%, 97%, or 99% identity at the amino acid level withtheir wild-type counterparts. Using the HBcAg having the amino acidsequence shown in SEQ ID NO:89 for illustration, the invention includesvaccine compositions comprising HBcAg polypeptides comprising, oralternatively consisting of, amino acid sequences which are at least80%, 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequenceshown in SEQ ID NO:89, and forms of this protein which have beenprocessed, where appropriate, to remove N-terminal leader sequence.These variants will generally be capable of associating to form dimericor multimeric structures. Methods which can be used to determine whetherproteins form such structures comprise gel filtration, agarose gelelectrophoresis, sucrose gradient centrifugation and electron microscopy(e.g., Koschel, M. et al., J. Virol 73: 2153-2160 (1999)).

Fragments of proteins which retain the ability to associate with eachother to form ordered and repetitive antigen or antigenic determinantarrays may comprise, or alternatively consist of, polypeptides which are15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids inlength. Examples of such protein fragments include fragments of proteinsdiscussed herein which are suitable for the preparation of coreparticles and/or non-natural molecular scaffolds.

Whether natural or non-natural, the core particle of the invention willgenerally have an organizer that is attached to the natural ornon-natural core particle by at least one covalent bond. The organizeris an element bound to a core particle in a non-random fashion thatprovides a nucleation site for creating an ordered and repetitiveantigen array. Ideally, but not necessarily, the organizer is associatedwith the core particle in a geometric order. Minimally, the organizercomprises a first attachment site.

In some embodiments of the invention, the ordered and repetitive arrayis formed by association between (1) either core particles ornon-natural molecular scaffolds and (2) either (a) an antigen orantigenic determinant or (b) one or more antigens or antigenicdeterminants. For example, bacterial pili or pilus-like structures areformed from proteins which are organized into ordered and repetitivestructures. Thus, in many instances, it will be possible to form orderedarrays of antigens or antigenic determinants by linking theseconstituents to bacterial pili or pilus-like structures either directlyor through an organizer.

As previously stated, the organizer may be any element comprising atleast one first attachment site that is bound to a core particle by atleast one covalent bond. An organizer may be a protein, a polypeptide, apeptide, an amino acid (i.e., a residue of a protein, a polypeptide orpeptide), a sugar, a polynucleotide, a natural or synthetic polymer, asecondary metabolite or compound (biotin, fluorescein, retinol,digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a combinationthereof, or a chemically reactive group thereof. In a more specificembodiment, the organizer may comprise a first attachment sitecomprising an antigen, an antibody or antibody fragment, biotin, avidin,strepavidin, a receptor, a receptor ligand, a ligand, a ligand-bindingprotein, an interacting leucine zipper polypeptide, an amino group, achemical group reactive to an amino group; a carboxyl group, chemicalgroup reactive to a carboxyl group, a sulfhydryl group, a chemical groupreactive to a sulfhydryl group, or a combination thereof

In one embodiment, the core particle of the non-natural molecularscaffold comprises a virus, a bacterial pilus, a structure formed frombacterial pilin, a bacteriophage, a virus-like particle, a viral capsidparticle or a recombinant form thereof. Any virus known in the arthaving an ordered and repetitive coat and/or core protein structure maybe selected as a non-natural molecular scaffold of the invention;examples of suitable viruses include sindbis and other alphaviruses,rhabdoviruses (e.g. vesicular stomatitis virus), picornaviruses (e.g.,human rhino virus, Aichi virus), togaviruses (e.g., rubella virus),orthomyxoviruses (e.g., Thogoto virus, Batken virus, fowl plague virus),polyomaviruses (e.g., polyomavirus BK, polyomavirus JC, avianpolyomavirus BFDV), parvoviruses, rotaviruses, bacteriophage Qβ,bacteriophage R17, bacteriophage M11, bacteriophage MX1, bacteriophageNL95, bacteriophage fr, bacteriophage GA, bacteriophage SP,bacteriophage MS2, bacteriophage f2, bacteriophage PP7, Norwalk virus,foot and mouth disease virus, a retrovirus, Hepatitis B virus, Tobaccomosaic virus, Flock House Virus, and human Papilomavirus (for example,see Table 1 in Bachman, M. F. and Zinkernagel, R. M., Immunol. Today17:553-558 (1996)).

In one embodiment, the invention utilizes genetic engineering of a virusto create a fusion between an ordered and repetitive viral envelopeprotein and an organizer comprising a heterologous protein, peptide,antigenic determinant or a reactive amino acid residue of choice. Othergenetic manipulations known to those in the art may be included in theconstruction of the non-natural molecular scaffold; for example, it maybe desirable to restrict the replication ability of the recombinantvirus through genetic mutation. The viral protein selected for fusion tothe organizer (i.e., first attachment site) protein should have anorganized and repetitive structure. Such an organized and repetitivestructure include paracrystalline organizations with a spacing of 5-15nm on the surface of the virus. The creation of this type of fusionprotein will result in multiple, ordered and repetitive organizers onthe surface of the virus. Thus, the ordered and repetitive organizationof the first attachment sites resulting therefrom will reflect thenormal organization of the native viral protein.

As will be discussed in more detail herein, in another embodiment of theinvention, the non-natural molecular scaffold is a recombinantalphavirus, and more specifically, a recombinant Sinbis virus.Alphaviruses are positive stranded RNA viruses that replicate theirgenomic RNA entirely in the cytoplasm of the infected cell and without aDNA intermediate (Strauss, J. and Strauss, E., Microbiol. Rev.58:491-562 (1994)). Several members of the alphavirus family, Sindbis(Xiong, C. et al., Science 243:1188-1191 (1989); Schlesinger, S., TrendsBiotechnol. 11:18-22 (1993)), Semliki Forest Virus (SFV) (Liljeström, P.& Garoff, H., Bio/Technology 9:1356-1361 (1991)) and others (Davis, N.L. et al., Virology 171:189-204 (1989)), have received considerableattention for use as virus-based expression vectors for a variety ofdifferent proteins (Lundstrom, K., Curr. Opin. Biotechnol. 8:578-582(1997); Liljeström, P., Curr. Opin. Biotechnol. 5:495-500 (1994)) and ascandidates for vaccine development. Recently, a number of patents haveissued directed to the use of alphaviruses for the expression ofheterologous proteins and the development of vaccines (see U.S. Pat.Nos. 5,766,602; 5,792,462; 5,739,026; 5,789,245 and 5,814,482). Theconstruction of the alphaviral scaffold of the invention may be done bymeans generally known in the art of recombinant DNA technology, asdescribed by the aforementioned articles, which are incorporated hereinby reference.

A variety of different recombinant host cells can be utilized to producea viral-based core particle for antigen or antigenic determinantattachment. For example, Alphaviruses are known to have a wide hostrange; Sindbis virus infects cultured mammalian, reptilian, andamphibian cells, as well as some insect cells (Clark, H., J. Natl.Cancer Inst. 51:645 (1973); Leake, C., J. Gen. Virol. 35:335 (1977);Stollar, V. in THE TOGAVIRUSES, R. W. Schlesinger, Ed., Academic Press,(1980), pp. 583-621). Thus, numerous recombinant host cells can be usedin the practice of the invention. BHK, COS, Vero, HeLa and CHO cells areparticularly suitable for the production of heterologous proteinsbecause they have the potential to glycosylate heterologous proteins ina manner similar to human cells (Watson, E. et al., Glycobiology 4:227,(1994)) and can be selected (Zang, M. et al., Bio/Technology 13:389(1995)) or genetically engineered (Renner W. et al., Biotech. Bioeng.4:476 (1995); Lee K. et al. Biotech. Bioeng. 50:336 (1996)) to grow inserum-free medium, as well as in suspension.

Introduction of the polynucleotide vectors into host cells can beeffected by methods described in standard laboratory manuals (see, e.g.,Sambrook, J. et al., eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd.edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989), Chapter 9; Ausubel, F. et al., eds., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997), Chapter 16),including methods such as electroporation, DEAE-dextran mediatedtransfection, transfection, microinjection, cationic lipid-mediatedtransfection, transduction, scrape loading, ballistic introduction, andinfection. Methods for the introduction of exogenous DNA sequences intohost cells are discussed in Felgner, P. et al., U.S. Pat. No. 5,580,859.

Packaged RNA sequences can also be used to infect host cells. Thesepackaged RNA sequences can be introduced to host cells by adding them tothe culture medium. For example, the preparation of non-infectivealpahviral particles is described in a number of sources, including“Sindbis Expression System”, Version C (Invitrogen Catalog No. K750-1).

When mammalian cells are used as recombinant host cells for theproduction of viral-based core particles, these cells will generally begrown in tissue culture. Methods for growing cells in culture are wellknown in the art (see, e.g., Celis, J., ed., CELL BIOLOGY, AcademicPress, 2^(nd) edition, (1998); Sambrook, J. et al., eds., MOLECULARCLONING, A LABORATORY MANUAL, 2^(nd). edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al.,eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc.(1997); Freshney, R., CULTURE OF ANIMAL CELLS, Alan R. Liss, Inc.(1983)).

As will be understood by those in the art, the first attachment site maybe or be a part of any suitable protein, polypeptide, sugar,polynucleotide, peptide (amino acid), natural or synthetic polymer, asecondary metabolite or combination thereof that may serve tospecifically attach the antigen or antigenic determinant of choice tothe non-natural molecular scaffold. In one embodiment, the attachmentsite is a protein or peptide that may be selected from those known inthe art. For example, the first attachment site may selected from thefollowing group: a ligand, a receptor, a lectin, avidin, streptavidin,biotin, an epitope such as an HA or T7 tag, Myc, Max, immunoglobulindomains and any other amino acid sequence known in the art that would beuseful as a first attachment site.

It should be further understood by those in the art that with anotherembodiment of the invention, the first attachment site may be createdsecondarily to the organizer (i.e., protein or polypeptide) utilized inconstructing the in-frame fusion to the capsid protein. For example, aprotein may be utilized for fusion to the envelope protein with an aminoacid sequence known to be glycosylated in a specific fashion, and thesugar moiety added as a result may then serve at the first attachmentsite of the viral scaffold by way of binding to a lectin serving as thesecondary attachment site of an antigen. Alternatively, the organizersequence may be biotinylated in vivo and the biotin moiety may serve asthe first attachment site of the invention, or the organizer sequencemay be subjected to chemical modification of distinct amino acidresidues in vitro, the modification serving as the first attachmentsite.

In another embodiment of the invention, the first attachment site isselected to be the JUN-FOS leucine zipper protein domain that is fusedin frame to the Hepatitis B capsid (core) protein (HBcAg). However, itwill be clear to all individuals in the art that other viral capsidproteins may be utilized in the fusion protein construct for locatingthe first attachment site in the non-natural molecular scaffold of theinvention.

In another embodiment of the invention, the first attachment site isselected to be a lysine or cysteine residue that is fused in frame tothe HBcAg. However, it will be clear to all individuals in the art thatother viral capsid or virus-like particles may be utilized in the fusionprotein construct for locating the first attachment in the non-naturalmolecular scaffold of the invention.

The JUN amino acid sequence utilized for the first attachment site isthe following:

(SEQ ID NO: 59) CGGRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVGC

-   -   In this instance, the anticipated second attachment site on the        antigen would be the FOS leucine zipper protein domain and the        amino acid sequence would be the following:

(SEQ ID NO: 60) CGGLTDTLQAETDQVEDEKSALQTEIANLLKEKEKLEFILAAHGGC

These sequences are derived from the transcription factors JUN and FOS,each flanked with a short sequence containing a cysteine residue on bothsides. These sequences are known to interact with each other. Theoriginal hypothetical structure proposed for the JUN-FOS dimer assumedthat the hydrophobic side chains of one monomer interdigitate with therespective side chains of the other monomer in a zipper-like manner(Landschulz et al., Science 240:1759-1764 (1988)). However, thishypothesis proved to be wrong, and these proteins are known to form ana-helical coiled coil (O'Shea et al., Science 243:538-542 (1989); O'Sheaet al., Cell 68:699-708 (1992); Cohen & Parry, Trends Biochem. Sci.11:245-248 (1986)). Thus, the term “leucine zipper” is frequently usedto refer to these protein domains for more historical than structuralreasons. Throughout this patent, the term “leucine zipper” is used torefer to the sequences depicted above or sequences essentially similarto the ones depicted above. The terms JUN and FOS are used for therespective leucine zipper domains rather than the entire JUN and FOSproteins.

As previously stated, the invention includes viral-based core particleswhich comprise, or alternatively consist of, a virus, virus-likeparticle, a phage, a viral capsid particle or a recombinant formthereof. Skilled artisans have the knowledge to produce such coreparticles and attach organizers thereto. The production of Hepatitis Bvirus-like particles and measles viral capsid particles as coreparticles is disclosed in Examples 17 to 22 of WO 00/32227, which isexplicitly incorporated by reference. In such embodiments, the JUNleucine zipper protein domain or FOS leucine zipper protein domain maybe used as an organizer, and hence as a first attachment site, for thenon-natural molecular scaffold of the invention.

Examples 23-29 provide details of the production of Hepatitis B coreparticles carrying an in-frame fused peptide with a reactive lysineresidue and antigens carrying a genetically fused cysteine residue, asfirst and second attachment site, respectively.

1 In other embodiments, the core particles used in compositions of theinvention are composed of a Hepatitis B capsid (core) protein (HBcAg), afragment of a HBcAg, or other protein or peptide which can form orderedarrays, which have been modified to either eliminate or reduce thenumber of free cysteine residues. Zhou et al. (J. Virol. 66:5393-5398(1992)) demonstrated that HBcAgs which have been modified to remove thenaturally resident cysteine residues retain the ability to associate andform multimeric structures. Thus, core particles suitable for use incompositions of the invention include those comprising modified HBcAgs,or fragments thereof, in which one or more of the naturally residentcysteine residues have been either deleted or substituted with anotheramino acid residue (e.g., a serine residue).2 The HBcAg is a protein generated by the processing of a Hepatitis Bcore antigen precursor protein. A number of isotypes of the HBcAg havebeen identified. For example, the HBcAg protein having the amino acidsequence shown in SEQ ID NO:132 is 183 amino acids in length and isgenerated by the processing of a 212 amino acid Hepatitis B core antigenprecursor protein. This processing results in the removal of 29 aminoacids from the N-terminus of the Hepatitis B core antigen precursorprotein. Similarly, the HBcAg protein having the amino acid sequenceshown in SEQ ID NO:134 is 185 amino acids in length and is generated bythe processing of a 214 amino acid Hepatitis B core antigen precursorprotein. The amino acid sequence shown in SEQ ID NO:134, as compared tothe amino acid sequence shown in SEQ ID NO:132, contains a two aminoacid insert at positions 152 and 153 in SEQ ID NO:134.

In most instances, vaccine compositions of the invention will beprepared using the processed form of a HBcAg (i.e., a HBcAg from whichthe N-terminal leader sequence (e.g., the first 29 amino acid residuesshown in SEQ ID NO:134) of the Hepatitis B core antigen precursorprotein have been removed).

Further, when HBcAgs are produced under conditions where processing willnot occur, the HBcAgs will generally be expressed in “processed” form.For example, bacterial systems, such as E. coli, generally do not removethe leader sequences, also referred to as “signal peptides,” of proteinswhich are normally expressed in eukaryotic cells. Thus, when an E. coliexpression system is used to produce HBcAgs of the invention, theseproteins will generally be expressed such that the N-terminal leadersequence of the Hepatitis B core antigen precursor protein is notpresent.

In one embodiment of the invention, a modified HBcAg comprising theamino acid sequence shown in SEQ ID NO:134, or subportion thereof, isused to prepare non-natural molecular scaffolds. In particular, modifiedHBcAgs suitable for use in the practice of the invention includeproteins in which one or more of the cysteine residues at positionscorresponding to positions 48, 61, 107 and 185 of a protein having theamino acid sequence shown in SEQ ID NO:134 have been either deleted orsubstituted with other amino acid residues (e.g., a serine residue). Asone skilled in the art would recognize, cysteine residues at similarlocations in HBcAg variants having amino acids sequences which differfrom that shown in SEQ ID NO:134 could also be deleted or substitutedwith other amino acid residues. The modified HBcAg variants can then beused to prepare vaccine compositions of the invention.

The present invention also includes HBcAg variants which have beenmodified to delete or substitute one or more additional cysteineresidues which are not found in polypeptides having the amino acidsequence shown in SEQ ID NO:134. Examples of such HBcAg variants havethe amino acid sequences shown in SEQ ID NOs:90 and 132. These variantcontain cysteines residues at a position corresponding to amino acidresidue 147 in SEQ ID NO:134. Thus, the vaccine compositions of theinvention include compositions comprising HBcAgs in which cysteineresidues not present in the amino acid sequence shown in SEQ ID NO:134have been deleted.

Under certain circumstances (e.g., when a heterobifunctionalcross-linking reagent is used to attach antigens or antigenicdeterminants to the non-natural molecular scaffold), the presence offree cysteine residues in the HBcAg is believed to lead to covalentcoupling of toxic components to core particles, as well as thecross-linking of monomers to form undefined species.

Further, in many instances, these toxic components may not be detectablewith assays performed on compositions of the invention. This is sobecause covalent coupling of toxic components to the non-naturalmolecular scaffold would result in the formation of a population ofdiverse species in which toxic components are linked to differentcysteine residues, or in some cases no cysteine residues, of the HBcAgs.In other words, each free cysteine residue of each HBcAg will not becovalently linked to toxic components. Further, in many instances, noneof the cysteine residues of particular HBcAgs will be linked to toxiccomponents. Thus, the presence of these toxic components may bedifficult to detect because they would be present in a mixed populationof molecules. The administration to an individual of HBcAg speciescontaining toxic components, however, could lead to a potentiallyserious adverse reaction.

It is well known in the art that free cysteine residues can be involvedin a number of chemical side reactions. These side reactions includedisulfide exchanges, reaction with chemical substances or metabolitesthat are, for example, injected or formed in a combination therapy withother substances, or direct oxidation and reaction with nucleotides uponexposure to UV light. Toxic adducts could thus be generated, especiallyconsidering the fact that HBcAgs have a strong tendency to bind nucleicacids. Detection of such toxic products in antigen-capsid conjugateswould be difficult using capsids prepared using HBcAgs containing freecysteines and heterobifunctional cross-linkers, since a distribution ofproducts with a broad range of molecular weight would be generated. Thetoxic adducts would thus be distributed between a multiplicity ofspecies, which individually may each be present at low concentration,but reach toxic levels when together.

In view of the above, one advantage to the use of HBcAgs in vaccinecompositions which have been modified to remove naturally residentcysteine residues is that sites to which toxic species can bind whenantigens or antigenic determinants are attached to the non-naturalmolecular scaffold would be reduced in number or eliminated altogether.Further, a high concentration of cross-linker can be used to producehighly decorated particles without the drawback of generating aplurality of undefined cross-linked species of HBcAg monomers (i.e., adiverse mixture of cross-linked monomeric HbcAgs).

A number of naturally occurring HBcAg variants suitable for use in thepractice of the present invention have been identified. Yuan et al., (J.Virol. 73:10122-10128 (1999)), for example, describe variants in whichthe isoleucine residue at position corresponding to position 97 in SEQID NO:134 is replaced with either a leucine residue or a phenylalanineresidue. The amino acid sequences of a number of HBcAg variants, as wellas several Hepatitis B core antigen precursor variants, are disclosed inGenBank reports AAF121240 (SEQ ID NO:89), AF121239 (SEQ ID NO:90),X85297 (SEQ ID NO:91), X02496 (SEQ ID NO:92), X85305 (SEQ ID NO:93),X85303 (SEQ ID NO:94), AF151735 (SEQ ID NO:95), X85259 (SEQ ID NO:96),X85286 (SEQ ID NO:97), X85260 (SEQ ID NO:98), X85317 (SEQ ID NO:99),X85298 (SEQ ID NO:100), AF043593 (SEQ ID NO:101), M20706 (SEQ IDNO:102), X85295 (SEQ ID NO:103), X80925 (SEQ ID NO:104), X85284 (SEQ IDNO:105), X85275 (SEQ ID NO:106), X72702 (SEQ ID NO:107), X85291 (SEQ IDNO:108), X65258 (SEQ ID NO:109), X85302 (SEQ ID NO:110), M32138 (SEQ IDNO:111), X85293 (SEQ ID NO:112), X85315 (SEQ ID NO:113), U95551 (SEQ IDNO:114), X85256 (SEQ ID NO:115), X85316 (SEQ ID NO:116), X85296 (SEQ IDNO:117), AB033559 (SEQ ID NO:118), X59795 (SEQ ID NO:119), X85299 (SEQID NO:120), X85307 (SEQ ID NO:121), X65257 (SEQ ID NO:122), X85311 (SEQID NO:123), X85301 (SEQ ID NO:124), X85314 (SEQ ID NO:125), X85287 (SEQID NO:126), X85272 (SEQ ID NO:127), X85319 (SEQ ID NO:128), AB010289(SEQ ID NO:129), X85285 (SEQ ID NO:130), AB010289 (SEQ ID NO:131),AF121242 (SEQ ID NO:132), M90520 (SEQ ID NO:135), P03153 (SEQ IDNO:136), AF110999 (SEQ ID NO:137), and M95589 (SEQ ID NO:138), thedisclosures of each of which are incorporated herein by reference. TheseHBcAg variants differ in amino acid sequence at a number of positions,including amino acid residues which corresponds to the amino acidresidues located at positions 12, 13, 21, 22, 24, 29, 32, 33, 35, 38,40, 42, 44, 45, 49, 51, 57, 58, 59, 64, 66, 67, 69, 74, 77, 80, 81, 87,92, 93, 97, 98, 100, 103, 105, 106, 109, 113, 116, 121, 126, 130, 133,135, 141, 147, 149, 157, 176, 178, 182 and 183 in SEQ ID NO:134.

HBcAgs suitable for use in the present invention may be derived from anyorganism so long as they are able to associate to form an ordered andrepetitive antigen array.

As noted above, generally processed HBcAgs (i.e., those which lackleader sequences) will be used in the vaccine compositions of theinvention. Thus, when HBcAgs having amino acid sequence shown in SEQ IDNOs:136, 137, or 138 are used to prepare vaccine compositions of theinvention, generally 30, 35-43, or 35-43 amino acid residues at theN-terminus, respectively, of each of these proteins will be omitted.

The present invention includes vaccine compositions, as well as methodsfor using these compositions, which employ the above described variantHBcAgs for the preparation of non-natural molecular scaffolds.

Further included within the scope of the invention are additional HBcAgvariants which are capable of associating to form dimeric or multimericstructures. Thus, the invention further includes vaccine compositionscomprising HBcAg polypeptides comprising, or alternatively consistingof, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97%, or99% identical to any of the amino acid sequences shown in SEQ IDNOs:89-132 and 134-138, and forms of these proteins which have beenprocessed, where appropriate, to remove the N-terminal leader sequence.

Whether the amino acid sequence of a polypeptide has an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, or 99% identical toone of the amino acid sequences shown in SEQ ID NOs:89-132 and 134-138,or a subportion thereof, can be determined conventionally using knowncomputer programs such the Bestfit program. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference amino acidsequence according to the present invention, the parameters are set suchthat the percentage of identity is calculated over the full length ofthe reference amino acid sequence and that gaps in homology of up to 5%of the total number of amino acid residues in the reference sequence areallowed.

The HBcAg variants and precursors having the amino acid sequences setout in SEQ ID NOs:89-132 and 134-136 are relatively similar to eachother. Thus, reference to an amino acid residue of a HBcAg variantlocated at a position which corresponds to a particular position in SEQID NO:134, refers to the amino acid residue which is present at thatposition in the amino acid sequence shown in SEQ ID NO:134. The homologybetween these HBcAg variants is for the most part high enough amongHepatitis B viruses that infect mammals so that one skilled in the artwould have little difficulty reviewing both the amino acid sequenceshown in SEQ ID NO:134 and that of a particular HBcAg variant andidentifying “corresponding” amino acid residues. For example, the HBcAgamino acid sequence shown in SEQ ID NO:135, which shows the amino acidsequence of a HBcAg derived from a virus which infect woodchucks, hasenough homology to the HBcAg having the amino acid sequence shown in SEQID NO:134 that it is readily apparent that a three amino acid residueinsert is present in SEQ ID NO:135 between amino acid residues 155 and156 of SEQ ID NO:134.

The HBcAgs of Hepatitis B viruses which infect snow geese and ducksdiffer enough from the amino acid sequences of HBcAgs of Hepatitis Bviruses which infect mammals that alignment of these forms of thisprotein with the amino acid sequence shown in SEQ ID NO:134 isdifficult. However, the invention includes vaccine compositions whichcomprise HBcAg variants of Hepatitis B viruses which infect birds, aswells as vaccine compositions which comprise fragments of these HBcAgvariants. HBcAg fragments suitable for use in preparing vaccinecompositions of the invention include compositions which containpolypeptide fragments comprising, or alternatively consisting of, aminoacid residues selected from the group consisting of 36-240, 36-269,44-240, 44-269, 36-305, and 44-305 of SEQ ID NO:137 or 36-240, 36-269,44-240, 44-269, 36-305, and 44-305 of SEQ ID NO:138. As one skilled inthe art would recognize, one, two, three or more of the cysteineresidues naturally present in these polypeptides (e.g., the cysteineresidues at position 153 is SEQ ID NO:137 or positions 34, 43, and 196in SEQ ID NO:138) could be either substituted with another amino acidresidue or deleted prior to their inclusion in vaccine compositions ofthe invention.

In one embodiment, the cysteine residues at positions 48 and 107 of aprotein having the amino acid sequence shown in SEQ ID NO:134 aredeleted or substituted with another amino acid residue but the cysteineat position 61 is left in place. Further, the modified polypeptide isthen used to prepare vaccine compositions of the invention.

As set out below in Example 31, the cysteine residues at positions 48and 107, which are accessible to solvent, may be removed, for example,by site-directed mutagenesis. Further, the inventors have found that theCys-48-Ser, Cys-107-Ser HBcAg double mutant constructed as described inExample 31 can be expressed in E. coli.

As discussed above, the elimination of free cysteine residues reducesthe number of sites where toxic components can bind to the HBcAg, andalso eliminates sites where cross-linking of lysine and cysteineresidues of the same or of neighboring HBcAg molecules can occur. Thecysteine at position 61, which is involved in dimer formation and formsa disulfide bridge with the cysteine at position 61 of another HBcAg,will normally be left intact for stabilization of HBcAg dimers andmultimers of the invention.

As shown in Example 32, cross-linking experiments performed with (1)HBcAgs containing free cysteine residues and (2) HBcAgs whose freecysteine residues have been made unreactive with iodacetamide, indicatethat free cysteine residues of the HBcAg are responsible forcross-linking between HBcAgs through reactions betweenheterobifunctional cross-linker derivatized lysine side chains, and freecysteine residues. Example 32 also indicates that cross-linking of HBcAgsubunits leads to the formation of high molecular weight species ofundefined size which cannot be resolved by SDS-polyacrylamide gelelectrophoresis.

When an antigen or antigenic determinant is linked to the non-naturalmolecular scaffold through a lysine residue, it may be advantageous toeither substitute or delete one or both of the naturally resident lysineresidues located at positions corresponding to positions 7 and 96 in SEQID NO:134, as well as other lysine residues present in HBcAg variants.The elimination of these lysine residues results in the removal ofbinding sites for antigens or antigenic determinants which could disruptthe ordered array and should improve the quality and uniformity of thefinal vaccine composition.

In many instances, when both of the naturally resident lysine residuesat positions corresponding to positions 7 and 96 in SEQ ID NO:134 areeliminated, another lysine will be introduced into the HBcAg as anattachment site for an antigen or antigenic determinant Methods forinserting such a lysine residue are set out, for example, in Example 23below. It will often be advantageous to introduce a lysine residue intothe HBcAg when, for example, both of the naturally resident lysineresidues at positions corresponding to positions 7 and 96 in SEQ IDNO:134 are altered and one seeks to attach the antigen or antigenicdeterminant to the non-natural molecular scaffold using aheterobifunctional cross-linking agent.

The C-terminus of the HBcAg has been shown to direct nuclearlocalization of this protein. (Eckhardt et al., J. Virol. 65:575-582(1991).) Further, this region of the protein is also believed to conferupon the HBcAg the ability to bind nucleic acids.

In some embodiments, vaccine compositions of the invention will containHBcAgs which have nucleic acid binding activity (e.g., which contain anaturally resident HBcAg nucleic acid binding domain). HBcAgs containingone or more nucleic acid binding domains are useful for preparingvaccine compositions which exhibit enhanced T-cell stimulatory activity.Thus, the vaccine compositions of the invention include compositionswhich contain HBcAgs having nucleic acid binding activity. Furtherincluded are vaccine compositions, as well as the use of suchcompositions in vaccination protocols, where HBcAgs are bound to nucleicacids. These HBcAgs may bind to the nucleic acids prior toadministration to an individual or may bind the nucleic acids afteradministration.

In other embodiments, vaccine compositions of the invention will containHBcAgs from which the C-terminal region (e.g., amino acid residues145-185 or 150-185 of SEQ ID NO:134) has been removed and do not bindnucleic acids. Thus, additional modified HBcAgs suitable for use in thepractice of the present invention include C-terminal truncation mutants.Suitable truncation mutants include HBcAgs where 1, 5, 10, 15, 20, 25,30, 34, 35, 36, 37, 38, 39 40, 41, 42 or 48 amino acids have beenremoved from the C-terminus

HBcAgs suitable for use in the practice of the present invention alsoinclude N-terminal truncation mutants. Suitable truncation mutantsinclude modified HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 aminoacids have been removed from the N-terminus

Further HBcAgs suitable for use in the practice of the present inventioninclude N- and C-terminal truncation mutants. Suitable truncationmutants include HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, and 17 aminoacids have been removed from the N-terminus and 1, 5, 10, 15, 20, 25,30, 34, 35, 36, 37, 38, 39 40, 41, 42 or 48 amino acids have beenremoved from the C-terminus

The invention further includes vaccine compositions comprising HBcAgpolypeptides comprising, or alternatively consisting of, amino acidsequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identicalto the above described truncation mutants.

As discussed above, in certain embodiments of the invention, a lysineresidue is introduced as a first attachment site into a polypeptidewhich forms the non-natural molecular scaffold. In preferredembodiments, vaccine compositions of the invention are prepared using aHBcAg comprising, or alternatively consisting of, amino acids 1-144 oramino acids 1-149 of SEQ ID NO:134 which is modified so that the aminoacids corresponding to positions 79 and 80 are replaced with a peptidehaving the amino acid sequence of Gly-Gly-Lys-Gly-Gly (SEQ ID NO:158)and the cysteine residues at positions 48 and 107 are either deleted orsubstituted with another amino acid residue, while the cysteine atposition 61 is left in place. The invention further includes vaccinecompositions comprising corresponding fragments of polypeptides havingamino acid sequences shown in any of SEQ ID NOs:89-132 and 135-136 whichalso have the above noted amino acid alterations.

The invention further includes vaccine compositions comprising fragmentsof a HBcAg comprising, or alternatively consisting of, an amino acidsequence other than that shown in SEQ ID NO:134 from which a cysteineresidue not present at corresponding location in SEQ ID NO:134 has beendeleted. One example of such a fragment would be a polypeptidecomprising, or alternatively consisting of, amino acids amino acids1-149 of SEQ ID NO:132 where the cysteine residue at position 147 hasbeen either substituted with another amino acid residue or deleted.

The invention further includes vaccine compositions comprising HBcAgpolypeptides comprising, or alternatively consisting of, amino acidsequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identicalto amino acids 1-144 or 1-149 of SEQ ID NO:134 and correspondingsubportions of a polypeptide comprising an amino acid sequence shown inany of SEQ ID NOs:89-132 or 134-136, as well as to amino acids 1-147 or1-152 of SEQ ID NO:158.

The invention also includes vaccine compositions comprising HBcAgpolypeptides comprising, or alternatively consisting of, amino acidsequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identicalto amino acids 36-240, 36-269, 44-240, 44-269, 36-305, and 44-305 of SEQID NO:137 or 36-240, 36-269, 44-240, 44-269, 36-305, and 44-305 of SEQID NO:138.

Vaccine compositions of the invention may comprise mixtures of differentHBcAgs. Thus, these vaccine compositions may be composed of HBcAgs whichdiffer in amino acid sequence. For example, vaccine compositions couldbe prepared comprising a “wild-type” HBcAg and a modified HBcAg in whichone or more amino acid residues have been altered (e.g., deleted,inserted or substituted). In most applications, however, only one typeof a HBcAg, or at least HBcAgs having essentially equivalent firstattachment sites, will be used because vaccines prepared using suchHBcAgs will present highly ordered and repetitive arrays of antigens orantigenic determinants. Further, preferred vaccine compositions of theinvention are those which present highly ordered and repetitive antigenarray

The invention further includes vaccine compositions where thenon-natural molecular scaffold is prepared using a HBcAg fused toanother protein. As discussed above, one example of such a fusionprotein is a HBcAg/FOS fusion. Other examples of HBcAg fusion proteinssuitable for use in vaccine compositions of the invention include fusionproteins where an amino acid sequence has been added which aids in theformation and/or stabilization of HBcAg dimers and multimers. Thisadditional amino acid sequence may be fused to either the N- orC-terminus of the HBcAg. One example, of such a fusion protein is afusion of a HBcAg with the GCN4 helix region of Saccharomyces cerevisiae(GenBank Accession No. P03069 (SEQ ID NO:154)).

The helix domain of the GCN4 protein forms homodimers via non-covalentinteractions which can be used to prepare and stabilize HBcAg dimers andmultimers.

In one embodiment, the invention provides vaccine compositions preparedusing HBcAg fusions proteins comprising a HBcAg, or fragment thereof,with a GCN4 polypeptide having the sequence of amino acid residues 227to 276 in SEQ ID NO:154 fused to the C-terminus. This GCN4 polypeptidemay also be fused to the N-terminus of the HbcAg.

HBcAg/src homology 3 (SH3) domain fusion proteins could also be used toprepare vaccine compositions of the invention. SH3 domains arerelatively small domains found in a number of proteins which confer theability to interact with specific proline-rich sequences in proteinbinding partners (see McPherson, Cell Signal 11:229-238 (1999).HBcAg/SH3 fusion proteins could be used in several ways. First, the SH3domain could form a first attachment site which interacts with a secondattachment site of the antigen or antigenic determinant. Similarly, aproline rich amino acid sequence could be added to the HBcAg and used asa first attachment site for an SH3 domain second attachment site of anantigen or antigenic determinant. Second, the SH3 domain could associatewith proline rich regions introduced into HBcAgs. Thus, SH3 domains andproline rich SH3 interaction sites could be inserted into either thesame or different HBcAgs and used to form and stabilized dimers andmultimers of the invention.

In other embodiments, a bacterial pilin, a subportion of a bacterialpilin, or a fusion protein which contains either a bacterial pilin orsubportion thereof is used to prepare vaccine compositions of theinvention. Examples of pilin proteins include pilins produced byEscherichia coli, Haemophilus influenzae, Neisseria meningitidis,Neisseria gonorrhoeae, Caulobacter crescentus, Pseudomonas stutzeri, andPseudomonas aeruginosa. The amino acid sequences of pilin proteinssuitable for use with the present invention include those set out inGenBank reports AJ000636 (SEQ ID NO:139), AJ132364 (SEQ ID NO:140),AF229646 (SEQ ID NO:141), AF051814 (SEQ ID NO:142), AF051815 (SEQ IDNO:143), and X00981 (SEQ ID NO:155), the entire disclosures of which areincorporated herein by reference.

Bacterial pilin proteins are generally processed to remove N-terminalleader sequences prior to export of the proteins into the bacterialperiplasm. Further, as one skilled in the art would recognize, bacterialpilin proteins used to prepare vaccine compositions of the inventionwill generally not have the naturally present leader sequence.

One specific example of a pilin protein suitable for use in the presentinvention is the P-pilin of E. coli (GenBank report AF237482 (SEQ IDNO:144)). An example of a Type-1 E. coli pilin suitable for use with theinvention is a pilin having the amino acid sequence set out in GenBankreport P04128 (SEQ ID NO:146), which is encoded by nucleic acid havingthe nucleotide sequence set out in GenBank report M27603 (SEQ IDNO:145). The entire disclosures of these GenBank reports areincorporated herein by reference. Again, the mature form of the abovereferenced protein would generally be used to prepare vaccinecompositions of the invention.

Bacterial pilins or pilin subportions suitable for use in the practiceof the present invention will generally be able to associate to formnon-natural molecular scaffolds.

Methods for preparing pili and pilus-like structures in vitro are knownin the art. Bullitt et al., Proc. Natl. Acad. Sci. USA 93:12890-12895(1996), for example, describe the in vitro reconstitution of E. coliP-pili subunits. Further, Eshdat et al., J. Bacteriol. 148:308-314(1981) describe methods suitable for dissociating Type-1 pili of E. coliand the reconstitution of pili. In brief, these methods are as follows:pili are dissociated by incubation at 37° C. in saturated guanidinehydrochloride. Pilin proteins are then purified by chromatography, afterwhich pilin dimers are formed by dialysis against 5 mMtris(hydroxymethyl) aminomethane hydrochloride (pH 8.0). Eshdat et al.also found that pilin dimers reassemble to form pili upon dialysisagainst the 5 mM tris(hydroxymethyl) aminomethane (pH 8.0) containing 5mM MgCl₂.

Further, using, for example, conventional genetic engineering andprotein modification methods, pilin proteins may be modified to containa first attachment site to which an antigen or antigenic determinant islinked through a second attachment site. Alternatively, antigens orantigenic determinants can be directly linked through a secondattachment site to amino acid residues which are naturally resident inthese proteins. These modified pilin proteins may then be used invaccine compositions of the invention.

Bacterial pilin proteins used to prepare vaccine compositions of theinvention may be modified in a manner similar to that described hereinfor HBcAg. For example, cysteine and lysine residues may be eitherdeleted or substituted with other amino acid residues and firstattachment sites may be added to these proteins. Further, pilin proteinsmay either be expressed in modified form or may be chemically modifiedafter expression. Similarly, intact pili may be harvested from bacteriaand then modified chemically.

In another embodiment, pili or pilus-like structures are harvested frombacteria (e.g., E. coli) and used to form vaccine compositions of theinvention. One example of pili suitable for preparing vaccinecompositions is the Type-1 pilus of E. coli, which is formed from pilinmonomers having the amino acid sequence set out in SEQ ID NO:146.

A number of methods for harvesting bacterial pili are known in the art.Bullitt and Makowski (Biophys. J. 74:623-632 (1998)), for example,describe a pilus purification method for harvesting P-pili from E. coli.According to this method, pili are sheared from hyperpiliated E. colicontaining a P-pilus plasmid and purified by cycles of solubilizationand MgCl₂ (1.0 M) precipitation. A similar purification method is setout below in Example 33.

Once harvested, pili or pilus-like structures may be modified in avariety of ways. For example, a first attachment site can be added tothe pili to which antigens or antigen determinants may be attachedthrough a second attachment site. In other words, bacterial pili orpilus-like structures can be harvested and modified to form non-naturalmolecular scaffolds.

Pili or pilus-like structures may also be modified by the attachment ofantigens or antigenic determinants in the absence of a non-naturalorganizer. For example, antigens or antigenic determinants could belinked to naturally occurring cysteine resides or lysine residues. Insuch instances, the high order and repetitiveness of a naturallyoccurring amino acid residue would guide the coupling of the antigens orantigenic determinants to the pili or pilus-like structures. Forexample, the pili or pilus-like structures could be linked to the secondattachment sites of the antigens or antigenic determinants using aheterobifunctional cross-linking agent.

When structures which are naturally synthesized by organisms (e.g.,pili) are used to prepare vaccine compositions of the invention, it willoften be advantageous to genetically engineer these organisms so thatthey produce structures having desirable characteristics. For example,when Type-1 pili of E. coli are used, the E. coli from which these piliare harvested may be modified so as to produce structures with specificcharacteristics. Examples of possible modifications of pilin proteinsinclude the insertion of one or more lysine residues, the deletion orsubstitution of one or more of the naturally resident lysine residues,and the deletion or substitution of one or more naturally residentcysteine residues (e.g., the cysteine residues at positions 44 and 84 inSEQ ID NO:146).

Further, additional modifications can be made to pilin genes whichresult in the expression products containing a first attachment siteother than a lysine residue (e.g., a FOS or JUN domain). Of course,suitable first attachment sites will generally be limited to those whichdo not prevent pilin proteins from forming pili or pilus-like structuressuitable for use in vaccine compositions of the invention.

Pilin genes which naturally reside in bacterial cells can be modified invivo (e.g., by homologous recombination) or pilin genes with particularcharacteristics can be inserted into these cells. For examples, pilingenes could be introduced into bacterial cells as a component of eithera replicable cloning vector or a vector which inserts into the bacterialchromosome. The inserted pilin genes may also be linked to expressionregulatory control sequences (e.g., a lac operator).

In most instances, the pili or pilus-like structures used in vaccinecompositions of the invention will be composed of single type of a pilinsubunit. Pili or pilus-like structures composed of identical subunitswill generally be used because they are expected to form structureswhich present highly ordered and repetitive antigen arrays.

However, the compositions of the invention also include vaccinescomprising pili or pilus-like structures formed from heterogenous pilinsubunits. The pilin subunits which form these pili or pilus-likestructures can be expressed from genes naturally resident in thebacterial cell or may be introduced into the cells. When a naturallyresident pilin gene and an introduced gene are both expressed in a cellwhich forms pili or pilus-like structures, the result will generally bestructures formed from a mixture of these pilin proteins. Further, whentwo or more pilin genes are expressed in a bacterial cell, the relativeexpression of each pilin gene will typically be the factor whichdetermines the ratio of the different pilin subunits in the pili orpilus-like structures.

When pili or pilus-like structures having a particular composition ofmixed pilin subunits is desired, the expression of at least one of thepilin genes can be regulated by a heterologous, inducible promoter. Suchpromoters, as well as other genetic elements, can be used to regulatethe relative amounts of different pilin subunits produced in thebacterial cell and, hence, the composition of the pili or pilus-likestructures.

In additional, while in most instances the antigen or antigenicdeterminant will be linked to bacterial pili or pilus-like structures bya bond which is not a peptide bond, bacterial cells which produce pilior pilus-like structures used in the compositions of the invention canbe genetically engineered to generate pilin proteins which are fused toan antigen or antigenic determinant. Such fusion proteins which formpili or pilus-like structures are suitable for use in vaccinecompositions of the invention.

As already discussed, viral capsids may be used for (1) the presentationor antigen or antigenic determinants and (2) the preparation of vaccinecompositions of the invention. Particularly, useful in the practice ofthe invention are viral capsid proteins, also referred to herein as“coat proteins,” which upon expression form capsids or capsid-likestructures. Thus, these capsid proteins can form core particles andnon-natural molecular scaffolds. Generally, these capsids or capsid-likestructures form ordered and repetitive arrays which can be used for thepresentation of antigens or antigenic determinants and the preparationof vaccine compositions of the invention.

One or more (e.g., one, two, three, four, five, etc.) antigens orantigenic determinants may be attached by any number of means to one ormore (e.g., one, two, three, four, five, etc.) proteins which form viralcapsids or capsid-like structures (e.g., bacteriophage coat proteins),as well as other proteins. For example, antigens or antigenicdeterminants may be attached to core particles using first and secondattachment sites. Further, one or more (e.g., one, two, three, four,five, etc.) heterobifunctional crosslinkers can be used to attachantigens or antigenic determinants to one or more proteins which formviral capsids or capsid-like structures.

Viral capsid proteins, or fragments thereof may be used, for example, toprepare core particles and vaccine compositions of the invention.Bacteriophage Qβ coat proteins, for example, can be expressedrecombinantly in E. coli. Further, upon such expression these proteinsspontaneously form capsids. Additionally, these capsids form ordered andrepetitive antigen or antigenic determinant arrays which can be used forantigen presentation and the preparation of vaccine compositions. Asdescribed below in Example 38, bacteriophage Qβ coat proteins can beused to prepare vaccine compositions which elicit immunologicalresponses to antigenic determinants.

Specific examples of bacteriophage coat proteins which can be used toprepare compositions of the invention include the coat proteins of RNAbacteriophages such as bacteriophage Qβ (SEQ ID NO:159; PIR Database,Accession No. VCBPQβ referring to Qβ CP and SEQ ID NO: 217; AccessionNo. AAA16663 referring to Qβ A1 protein), bacteriophage R17 (SEQ IDNO:160; PIR Accession No. VCBPR7), bacteriophage fr (SEQ ID NO:161; PIRAccession No. VCBPFR), bacteriophage GA (SEQ ID NO:162; GenBankAccession No. NP-040754), bacteriophage SP (SEQ ID NO:163; GenBankAccession No. CAA30374 referring to SP CP and SEQ ID NO: 254; AccessionNo. referring to SP A1 protein), bacteriophage MS2 (SEQ ID NO:164; PIRAccession No. VCBPM2), bacteriophage M11 (SEQ ID NO:165; GenBankAccession No. AAC06250), bacteriophage MX1 (SEQ ID NO:166; GenBankAccession No. AAC14699), bacteriophage NL95 (SEQ ID NO:167; GenBankAccession No. AAC14704), bacteriophage f2 (SEQ ID NO: 215; GenBankAccession No. P03611), bacteriophage PP7 (SEQ ID NO: 253), As oneskilled in the art would recognize, any protein which forms capsids orcapsid-like structures can be used for the preparation of vaccinecompositions of the invention. Furthermore, the A1 protein ofbacteriophage Qβ or C-terminal truncated forms missing as much as 100,150 or 180 amino acids from its C-terminus may be incorporated in acapsid assembly of Qβ coat proteins. The A1 protein may also be fused toan organizer and hence a first attachment site, for attachment ofAntigens containing a second attachment site. Generally, the percentageof A1 protein relative to Qβ CP in the capsid assembly will be limited,in order to insure capsid formation. A1 protein accession No. AAA16663(SEQ ID NO: 217).

Qβ coat protein has also been found to self-assemble into capsids whenexpressed in E. coli (Kozlovska T M. et al., GENE 137: 133-137 (1993)).The obtained capsids or virus-like particles showed an icosahedralphage-like capsid structure with a diameter of 25 nm and T=3 quasisymmetry. Further, the crystal structure of phage Qβ has been solved.The capsid contains 180 copies of the coat protein, which are linked incovalent pentamers and hexamers by disulfide bridges (Golmohammadi, R.et al., Structure 4: 543-5554 (1996)). Other RNA phage coat proteinshave also been shown to self-assemble upon expression in a bacterialhost (Kastelein, R A. et al., Gene 23: 245-254 (1983), Kozlovskaya, T M.et al., Dokl. Akad. Nauk SSSR 287: 452-455 (1986), Adhin, M R. et al.,Virology 170: 238-242 (1989), Ni, C Z., et al., Protein Sci. 5:2485-2493 (1996), Priano, C. et al., J. Mol. Biol. 249: 283-297 (1995)).The Qβ phage capsid contains, in addition to the coat protein, the socalled read-through protein A1 and the maturation protein A2. A1 isgenerated by suppression at the UGA stop codon and has a length of 329aa. The capsid of phage Qβ recombinant coat protein used in theinvention is devoid of the A2 lysis protein, and contains RNA from thehost. The coat protein of RNA phages is an RNA binding protein, andinteracts with the stem loop of the ribosomal binding site of thereplicase gene acting as a translational repressor during the life cycleof the virus. The sequence and structural elements of the interactionare known (Witherell, G W. & Uhlenbeck, O C. Biochemistry 28: 71-76(1989); Lim F. et al., J. Biol. Chem. 271: 31839-31845 (1996)). The stemloop and RNA in general are known to be involved in the virus assembly(Golmohammadi, R. et al., Structure 4: 543-5554 (1996))

Proteins or protein domains may affect the structure and assembly of theparticle even more then a short peptide. As an example, proper foldingof antigens comprising disulfide bridges will generally not be possiblein the cytoplasm of E. coli, where the Qβ particles are expressed.Likewise, glycosylation is generally not possible in prokaryoticexpression systems. It is therefore an advantage of the contemplatedinvention described here to attach the antigen to the particle bystarting with the already assembled particle and the isolated antigen.This allows expression of both the particle and the antigen in anexpression host guaranteeing proper folding of the antigen, and properfolding and assembly of the particle.

It is a finding of this invention, that one or several antigen moleculesmay be attached to one subunit of the capsid of RNA phages coatproteins. A specific feature of the capsid of the coat protein of RNAphages and in particular of Qβ capsid is thus the possibility to coupleseveral antigens per subunit. This allows for the generation of a denseantigen array. Other viral capsids used for covalent attachment ofantigens by way of chemical cross-linking, such for example a HBcAgmodified with a lysine residue in its major immunodominant region (MIR;WO 00/32227), show coupling density of maximally 0.5 antigens persubunit. The distance between the spikes (corresponding to the MIR) ofHBcAg is 50 A (Wynne, S A. et al., Mol. Cell 3: 771-780 (1999)), andtherefore an antigen array with distances shorter than 50 A cannot begenerated

Capsids of Qβ coat protein display a defined number of lysine residueson their surface, with a defined topology with three lysine residuespointing towards the interior of the capsid and interacting with theRNA, and four other lysine residues exposed to the exterior of thecapsid. These defined properties favor the attachment of antigens to theexterior of the particle, and not to the interior where the lysineresidues interact with RNA. Capsids of other RNA phage coat proteinsalso have a defined number of lysine residues on their surface and adefined topology of these lysine residues. Another advantage of thecapsids derived from RNA phages is their high expression yield inbacteria, that allows to produce large quantities of material ataffordable cost.

Another feature of the capsid of Qβ coat protein is its stability. Qβsubunits are bound via disulfide bridges to each other, covalentlylinking the subunits. Qβ capsid protein also shows unusual resistance toorganic solvents and denaturing agents. Surprisingly, we have observedthat DMSO and acetonitrile concentrations as high as 30%, andGuanidinium concentrations as high as 1 M could be used withoutaffecting the stability or the ability to form antigen arrays of thecapsid. Thus, theses organic solvents may be used to couple hydrophobicpeptides. The high stability of the capsid of Qβ coat protein is animportant feature pertaining to its use for immunization and vaccinationof mammals and humans in particular. The resistance of the capsid toorganic solvent allows the coupling of antigens not soluble in aqueousbuffers.

Insertion of a cysteine residue into the N-terminal β-hairpin of thecoat protein of the RNA phage MS-2 has been described in the U.S. Pat.No. 5,698,424. We note however, that the presence of an exposed freecysteine residue in the capsid may lead to oligomerization of capsids byway of disulfide bridge formation. Other attachments contemplated inU.S. Pat. No. 5,698,424 involve the formation of disulfide bridgesbetween the antigen and the Qβ particle. Such attachments are labile tosulfhydryl-moiety containing molecules.

The reaction between an initial disulfide bridge formed with acys-residue on Qβ, and the antigen containing a free sulfhydryl residuereleases sulfhydryl containing species other than the antigen. Thesenewly formed sulfhydryl containing species can react again with otherdisulfide bridges present on the particle, thus establishing anequilibrium. Upon reaction with the disulfide bridge formed on theparticle, the antigen may either form a disulfide bridge with thecys-residue from the particle, or with the cys-residue of the leavinggroup molecule which was forming the initial disulfide bridge on theparticle. Moreover, the other method of attachment described, using ahetero-bifunctional cross-linker reacting with a cysteine on the Qβparticle on one side, and with a lysine residue on the antigen on theother side, leads to a random orientation of the antigens on theparticle.

We further note that, in contrast to the capsid of the Qβ and Fr coatproteins, recombinant MS-2 described in patent application U.S. Pat. No.5,698,424 is essentially free of nucleic acids, while RNA is packagedinside the two capsids mentioned above.

We describe new and inventive compositions allowing the formation ofrobust antigen arrays with variable antigen density. We show that muchhigher epitope density can be achieved than usually obtained with otherVLPs. We also disclose compositions with simultaneous display of severalantigens with appropriate spacing, and compositions wherein the additionof accessory molecules, enhancing solubility or modifying the capsid ina suitable and desired way.

The preparation of compositions of capsids of RNA phage coat proteinswith a high epitope density is disclosed in this application. As askilled artisan in the Art would know, the conditions for the assemblyof the ordered and repetitive antigen array depend for a good part onthe antigen and on the selection of a second attachment site on theantigen. In the case of the absence of a useful second attachment site,such a second attachment has to be engineered to the antigen.

A prerequisite in designing a second attachment site, is the choice ofthe position at which it should be fused, inserted or generallyengineered. A skilled artisan would know how to find guidance inselecting the position of the second attachment site. A crystalstructure of the antigen may provide information on the availability ofthe C- or N-termini of the molecule (determined for example from theiraccessibility to solvent), or on the exposure to solvent of residuessuitable for use as second attachment sites, such as cysteine residues.Exposed disulfide bridges, as is the case for Fab fragments, may also bea source of a second attachment site, since they can be generallyconverted to single cysteine residues through mild reduction. Ingeneral, in the case where immunization with a self-antigen is aiming atinhibiting the interaction of this self-antigen with its naturalligands, the second attachment site will be added such that it allowsgeneration of antibodies against the site of interaction with thenatural ligands. Thus, the location of the second attachment site willselected such, that steric hindrance from the second attachment site orany amino acid linker containing it, is avoided. In further embodiments,an antibody response directed at a site distinct from the interactionsite of the self-antigen with its natural ligand is desired. In suchembodiments, the second attachment site may be selected such that itprevents generation of antibodies against the interaction site of theself-antigen with its natural ligands.

Other criteria in selecting the position of the second attachment siteinclude the oligomerization state of the antigen, the site ofoligomerization, the presence of a cofactor, and the availability ofexperimental evidence disclosing sites in the antigen structure andsequence where modification of the antigen is compatible with thefunction of the self-antigen, or with the generation of antibodiesrecognizing the self-antigen.

In some embodiments, engineering of a second attachment site onto theantigen requires the fusion of an amino acid linker containing an aminoacid suitable as second attachment site according to the disclosures ofthis invention. In a preferred embodiment, the amino acid is cysteine.The selection of the amino acid linker will be dependent on the natureof the self-antigen, on its biochemical properties, such as pI, chargedistribution, glycosylation. In general, flexible amino acid linkers arefavored. Examples of amino acid linkers are the hinge region ofImmunoglobulins, glycine serine linkers (GGGGS)_(n) (SEQ ID NO:407), andglycine linkers (G)_(n) all further containing a cysteine residue assecond attachment site and optionally further glycine residues. (In thefollowing are examples of said amino acid linkers:

N-terminal gamma1: (SEQ ID NO: 408) CGDKTHTSPP C-terminal gamma 1:(SEQ ID NO: 409) DKTHTSPPCG N-terminal gamma 3: (SEQ ID NO: 410)CGGPKPSTPPGSSGGAP C-terminal gamma 3: (SEQ ID NO: 411)PKPSTPPGSSGGAPGGCG N-terminal glycine linker: (SEQ ID NO: 412) GCGGGGC-terminal glycine linker: (SEQ ID NO: 413) GGGGCG

For peptides, GGCG (SEQ ID NO:414) linkers at the C-terminus of thepeptide, or CGG at its N-terminus have shown to be useful. In general,glycine residues will be inserted between bulky amino acids and thecysteine to be used as second attachment site.

A particularly favored method of attachment of antigens to VLPs, and inparticular to capsids of RNA phage coat proteins is the linking of alysine residue on the surface of the capsid of RNA phage coat proteinswith a cysteine residue on the antigen. To be effective as secondattachment site, a sulfhydryl group must be available for coupling.Thus, a cysteine residue has to be in its reduced state, that is a freecysteine or a cysteine residue with a free sulfhydryl group has to beavailable. In the instant where the cysteine residue to function assecond attachment site is in an oxidized form, for example if it isforming a disulfide bridge, reduction of this disulfide bridge with e.g.DTT, TCEP or β-mercaptoethanol is required.

It is a finding of this application that epitope density on the capsidof RNA phage coat proteins can be modulated by the choice ofcross-linker and other reaction conditions. For example, thecross-linkers Sulfo-GMBS and SMPH allow reaching higher epitope densitythan the cross-linker Sulfo-MBS under the same reaction conditions.Derivatization is positively influenced by high concentration ofreactands, and manipulation of the reaction conditions can be used tocontrol the number of antigens coupled to RNA phages capsid proteins,and in particular to Qβ capsid protein.

From theoretical calculation, the maximally achievable number ofglobular protein antigens of a size of 17 kDa does not exceed 0.5. Thus,several of the lysine residues of the capsid of Qβ coat protein will bederivatized with a cross-linker molecule, yet be devoid of antigen. Thisleads to the disappearance of a positive charge, which may bedetrimental to the solubility and stability of the conjugate. Byreplacing some of the lysine residues with arginines, such is the casein the disclosed Qβ coat protein mutant, we prevent the excessivedisappearance of positive charges since the arginine residues do notreact with the cross-linker.

In further embodiments, we disclose a Qβ mutant coat protein withadditional lysine residues, suitable for obtaining high density arraysof antigens.

The crystal structure of several RNA bacteriophages has been determined(Golmohammadi, R. et al., Structure 4:543-554 (1996)). Using suchinformation, one skilled in the art could readily identify surfaceexposed residues and modify bacteriophage coat proteins such that one ormore reactive amino acid residues can be inserted. Thus, one skilled inthe art could readily generate and identify modified forms ofbacteriophage coat proteins which can be used in the practice of theinvention. Thus, variants of proteins which form capsids or capsid-likestructures (e.g., coat proteins of bacteriophage Qβ, bacteriophage R17,bacteriophage fr, bacteriophage GA, bacteriophage SP, and bacteriophageMS2) can also be used to prepare vaccine compositions of the invention.

Although the sequence of the variants proteins discussed above willdiffer from their wild-type counterparts, these variant proteins willgenerally retain the ability to form capsids or capsid-like structures.Thus, the invention further includes vaccine compositions which containvariants of proteins which form capsids or capsid-like structures, aswell as methods for preparing such vaccine compositions, individualprotein subunits used to prepare such vaccine compositions, and nucleicacid molecules which encode these protein subunits. Thus, includedwithin the scope of the invention are variant forms of wild-typeproteins which form ordered and repetitive antigen arrays (e.g.,variants of proteins which form capsids or capsid-like structures) andretain the ability to associate and form capsids or capsid-likestructures.

As a result, the invention further includes vaccine compositionscomprising proteins comprising, or alternatively consisting of, aminoacid sequences which are at least 80%, 85%, 90%, 95%, 97%, or 99%identical to wild-type proteins which form ordered arrays. In manyinstances, these proteins will be processed to remove signal peptides(e.g., heterologous signal peptides).

Further included within the scope of the invention are nucleic acidmolecules which encode proteins used to prepare vaccine compositions ofthe invention.

In particular embodiments, the invention further includes vaccinecompositions comprising proteins comprising, or alternatively consistingof, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97%, or99% identical to any of the amino acid sequences shown in SEQ IDNOs:159-167, and forms of these proteins which have been processed,where appropriate, to remove the N-terminal leader sequence.

Proteins suitable for use in the practice of the present invention alsoinclude C-terminal truncation mutants of proteins which form capsids orcapsid-like structures, as well as other ordered arrays. Specificexamples of such truncation mutants include proteins having an aminoacid sequence shown in any of SEQ ID NOs:159-167 where 1, 2, 5, 7, 9,10, 12, 14, 15, or 17 amino acids have been removed from the C-terminusNormally, C-terminal truncation mutants used in the practice of theinvention will retain the ability to form capsids or capsid-likestructures.

Further proteins suitable for use in the practice of the presentinvention also include N-terminal truncation mutants of proteins whichform capsids or capsid-like structures. Specific examples of suchtruncation mutants include proteins having an amino acid sequence shownin any of SEQ ID NOs:159-167 where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17amino acids have been removed from the N-terminus Normally, N-terminaltruncation mutants used in the practice of the invention will retain theability to form capsids or capsid-like structures.

Additional proteins suitable for use in the practice of the presentinvention include — and C-terminal truncation mutants which form capsidsor capsid-like structures. Suitable truncation mutants include proteinshaving an amino acid sequence shown in any of SEQ ID NOs:159-167 where1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids have been removed fromthe N-terminus and 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids havebeen removed from the C-terminus Normally, N-terminal and C-terminaltruncation mutants used in the practice of the invention will retain theability to form capsids or capsid-like structures.

The invention further includes vaccine compositions comprising proteinscomprising, or alternatively consisting of, amino acid sequences whichare at least 80%, 85%, 90%, 95%, 97%, or 99% identical to the abovedescribed truncation mutants.

The invention thus includes vaccine compositions prepared from proteinswhich form ordered arrays, methods for preparing vaccine compositionsfrom individual protein subunits, methods for preparing these individualprotein subunits, nucleic acid molecules which encode these subunits,and methods for vaccinating and/or eliciting immunological responses inindividuals using vaccine compositions of the invention.

B. Construction of an Antigen or Antigenic Determinant with a SecondAttachment Site

The second element in the compositions of the invention is an antigen orantigenic determinant possessing at least one second attachment sitecapable of association through at least one non-peptide bond to thefirst attachment site of the non-natural molecular scaffold. Theinvention provides for compositions that vary according to the antigenor antigenic determinant selected in consideration of the desiredtherapeutic effect. Other compositions are provided by varying themolecule selected for the second attachment site.

However, when bacterial pili, or pilus-like structures, pilin proteinsare used to prepare vaccine compositions of the invention, antigens orantigenic determinants may be attached to pilin proteins by theexpression of pilin/antigen fusion proteins. Similarly, when proteinsother than pilin proteins (e.g., viral capsid proteins) are used toprepare vaccine compositions of the invention, antigens or antigenicdeterminants may be attached to these non-pilin proteins by theexpression of non-pilin protein/antigen fusion proteins. Antigens orantigenic determinants may also be attached to bacterial pili,pilus-like structures, pilin proteins, and other proteins which formordered arrays through non-peptide bonds.

Antigens of the invention may be selected from the group consisting ofthe following: (a) proteins suited to induce an immune response againstcancer cells; (b) proteins suited to induce an immune response againstinfectious diseases; (c) proteins suited to induce an immune responseagainst allergens, (d) proteins suited to induce an immune response infarm animals, and (e) fragments (e.g., a domain) of any of the proteinsset out in (a)-(d).

In one specific embodiment of the invention, the antigen or antigenicdeterminant is one that is useful for the prevention of infectiousdisease. Such treatment will be useful to treat a wide variety ofinfectious diseases affecting a wide range of hosts, e.g., human, cow,sheep, pig, dog, cat, other mammalian species and non-mammalian speciesas well. Treatable infectious diseases are well known to those skilledin the art, examples include infections of viral etiology such as HIV,influenza, Herpes, viral hepatitis, Epstein Bar, polio, viralencephalitis, measles, chicken pox, etc.; or infections of bacterialetiology such as pneumonia, tuberculosis, syphilis, etc.; or infectionsof parasitic etiology such as malaria, trypanosomiasis, leishmaniasis,trichomoniasis, amoebiasis, etc. Thus, antigens or antigenicdeterminants selected for the compositions of the invention will be wellknown to those in the medical art; examples of antigens or antigenicdeterminants include the following: the HIV antigens gp140 and gp160;the influenza antigens hemagglutinin, M2 protein and neuraminidase,Hepatitis B surface antigen, circumsporozoite protein of malaria.

In specific embodiments, the invention provides vaccine compositionssuitable for use in methods for preventing and/or attenuating diseasesor conditions which are caused or exacerbated by “self” gene products(e.g., tumor necrosis factors). Thus, vaccine compositions of theinvention include compositions which lead to the production ofantibodies that prevent and/or attenuate diseases or conditions causedor exacerbated by “self” gene products. Examples of such diseases orconditions include graft versus host disease, IgE-mediated allergicreactions, anaphylaxis, adult respiratory distress syndrome, Crohn'sdisease, allergic asthma, acute lymphoblastic leukemia (ALL),non-Hodgkin's lymphoma (NHL), Graves' disease, systemic lupuserythematosus (SLE), inflammatory autoimmune diseases, myastheniagravis, immunoproliferative disease lymphadenopathy (IPL),angioimmunoproliferative lymphadenopathy (AIL), immunoblastivelymphadenopathy (IBL), rheumatoid arthritis, diabetes, multiplesclerosis, Alzheimer disease and osteoporosis.

In related specific embodiments, compositions of the invention are animmunotherapeutic that may be used for the treatment of allergies orcancer.

The selection of antigens or antigenic determinants for the preparationof compositions and for use in methods of treatment for allergies wouldbe known to those skilled in the medical arts treating such disorders.Representative examples of such antigens or antigenic determinantsinclude the following: bee venom phospholipase A₂, Bet v I (birch pollenallergen), 5 Dol m V (white-faced hornet venom allergen), Mellitin andDer p I (House dust mite allergen), as well as fragments of each whichcan be used to elicit immunological responses.

As indicated, a preferred antigen or antigenic determinant is Der p I.Der p I is a 25 kD protease found in house dust mite faecal particles.Der p I represents the major allergic molecule of house dust mite.Accordingly, 80% of mite allergic patients have anti-Der p I IgEantibodies. In particular, the peptides p52-72 and p117-133, amongothers, are known to comprise epitopes, which are recognized byantibodies specific for the native Der p I. IgE antibodies raised in apolyclonal response to the whole antigen bind with high affinity to thepeptide region 59-94 (L. Pierson-Mullany et al. (2000) MolecularImmunology). Other regions also bind IgE with high affinity. The peptidep117-133 contains a free cysteine at its N-terminus, preferablyrepresenting the second attachment site in accordance with theinvention. 3D model assigns peptides p52-72 and p117-133 to the surfaceof the whole protein. However, other fragments of the Der p I proteinmay comprise B cell epitopes being preferably suitable for the presentinvention.

The selection of antigens or antigenic determinants for compositions andmethods of treatment for cancer would be known to those skilled in themedical arts treating such disorders. Representative examples of suchtypes of antigens or antigenic determinants include the following: Her2(breast cancer), GD2 (neuroblastoma), EGF-R (malignant glioblastoma),CEA (medullary thyroid cancer), and CD52 (leukemia), human melanomaprotein gp100, human melanoma protein melan-A/MART-1, tyrosinase, NA17-Ant protein, MAGE-3 protein, p53 protein, HPV16 E7 protein, as well asfragments of each which can be used to elicit immunological responses.Further preferred antigenic determinants useful for compositions andmethods of treatment for cancer are molecules and antigenic determinantsinvolved in angiogenesis. Angiogenesis, the formation of new bloodvessels, plays an essential role in physiological and pathophysiologicalprocesses such as wound healing and solid tumor growth, respectively(Folkman, J. (1995) Nat. medicine 1, 27-31; Folkman, J., and Klagsbrun,M. (1987) Science 235, 442-446; Martiny-Baron, G., and Marmé, D. (1995)Curr. Opin. Biotechnol. 6, 675-680; Risau, W. (1997) Nature 386,671-674). Rapidly growing tumors initiate and depend on the formation ofblood vessels to provide the required blood supply. Thus, antiangiogenicagents might be effective as an anticancer therapy.

Among several putative angiogenic factors that have been identified sofar vascular endothelial growth factor (VEGF) is a potent endothelialcell specific mitogen and a primary stimulant of the vascularization ofmany solid tumors. Although recent findings implicate that a set ofangiogenic factors must be perfectly orchestrated to form functionalvessels, it seems that the blockade of even a single growth factor mightlimit disease-induced vascular growth. Thus, blockade of VEGF may be apremium target for intervention in tumor induced angiogenesis. To targetthe endothelium rather than the tumor itself has recently emerged as anovel strategy to fight tumors (Millauer, B., Shawver, L. K., Plate, K.H., Risau, W., and Ullrich, A. (1994) Nature 367, 576-579; Kim, J., Li,B., Winer, J., Armanini, M., Gillett, N., Phillip, H. S., Ferrara, N.(1993) Nature 362, 841-844). In contrast to tumors, which easily mutatetarget structures recognized by the immune system, endothelial cells donot usually escape the immune system or other therapeutic regimens.

An anti-VEGFR-II antibody (IMC-1C11) and an anti-VEGF antibody have beendisclosed (Lu, D., Kussie, P., Pytowski, B., Persaud, K., Bohlen, P.,Witte, L., Zhu, Z. (2000) J. Biol. Chem. 275, 14321-14330; Presta, L. G,Chen, H., O'Connor, S J., Chisholm, V., Meng, Y G., Krummen, L.,Winkler, M., Ferrara N. (1997) Cancer Res. 47, 4593-4599). The formerneutralizing monoclonal anti-VEGFR-2 antibody recognizes an epitope thathas been identified as putative VEGF/VEGFR-II binding site (Piossek, C.,Schneider-Mergener, J., Schirner, M., Vakalopoulou, E., Germeroth, L.,Thierauch, K. H. (1999) J Biol Chem. 274, 5612-5619).

Thus, in another preferred embodiment of the invention, the antigen orantigenic determinant is a peptide derived from the VEGFR-II contactsite. This provides a composition and a vaccine composition inaccordance with the invention, which may have antiangiogenic propertiesuseful for the treatment of cancer. Inhibition of tumor growth in miceusing sera specific for VEGFR-2 has been demonstrated (Wei, Y Q., Wang,Q R., Zhao, X., Yang, L., Tian, L., Lu, Y., Kang, B., Lu, C J., Huang, MJ., Lou, Y Y., Xiao, F., He, Q M., Shu, J M., Xie, X J., Mao, Y Q., Lei,S., Luo, F., Zhou, L Q., Liu, C E., Zhou, H., Jiang, Y., Peng, F., Yuan,L P., Li, Q., Wu, Y., Liu, J Y. (2000) Nature Medicine 6, 1160-1165).Therefore, further preferred antigenic determinants suitable forinventive compositions and antiangiogenic vaccine compositions inaccordance with the invention comprise either the human VEGFR-II derivedpeptide with the sequence CTARTELNVGIDFNWEYPSSKHQHKK (SEQ ID NO:351),and/or the murine VEGFR-II derived peptide having the sequenceCTARTELNVGLDFTWHSPPSKSHHKK (SEQ ID NO:352), and/or the relevantextracellular globular domains 1-3 of the VEGFR-II.

Therefore, in a preferred embodiment of the invention, the vaccinecomposition comprises a core particle selected from a virus-likeparticle or a bacterial pilus and a VEGFR-II derived peptide or afragment thereof as an antigen or antigenic determinant in accordancewith the present invention.

The selection of antigens or antigenic determinants for compositions andmethods of treatment for other diseases or conditions associated withself antigens would be also known to those skilled in the medical artstreating such disorders. Representative examples of such antigens orantigenic determinants are, for example, lymphotoxins (e.g. Lymphotoxinα (LT α), Lymphotoxin β (LT β)), and lymphotoxin receptors, Receptoractivator of nuclear factor kB ligand (RANKL), vascular endothelialgrowth factor (VEGF), vascular endothelial growth factor receptor(VEGF-R), Interleukin-5, Interleukin-17, Interleukin-13, CCL21, CXCL12,SDF-1, MCP-1, Endoglin, Resistin, GHRH, LHRH, TRH, MIF, Eotaxin,Bradykinin, BLC, Tumor Necrosis Factor α and amyloid beta peptide(Aβ₁₋₄₂) (SEQ ID NO: 220), as well as fragments of each which can beused to elicit immunological responses. In a preferred embodiment, theantigen is the amyloid beta peptide (Aβ₁₋₄₂) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 220), or a fragment thereof. Theamyloid beta protein is SEQ ID NO: 218. The amyloid beta precursorprotein is SEQ ID NO: 219.

In another preferred embodiment of the invention, the antigen orantigenic determinant is an angiotensin peptide or a fragment thereof.The term “angiotensin peptide” as used herein, shall encompass anypeptide comprising the sequence, or fragments thereof, ofangiotensinogen, angiotensin I or angiotensin II. The sequences are asfollows: Angiotensinogen: DRVYIHPFHLVIHN (SEQ ID NO:353); Angiotensin I:DRVYIHPFHL (SEQ ID NO:354); Angiotensin II: DRVYIHPF (SEQ ID NO:355).Typically, one or more additional amino acids are added either at the C-or at the N-terminus of the angiotensin peptide sequences. The sequenceof the angiotensin peptides corresponds to the human sequence, which isidentical to the murine sequence. Therefore, immunization of a human ora mouse with vaccines or compositions, respectively, comprising suchangiotensin peptides as antigenic determinant in accordance with theinvention, is a vaccination against a self-antigen. Those additionalamino acids are, in particular, valuable for an oriented and orderedassociation to the core particle.

Preferably, the angiotensin peptide has an amino acid sequence selectedfrom the group consisting of a) the amino acid sequence of CGGDRVYIHPF(SEQ ID NO:380); b) the amino acid sequence of CGGDRVYIHPFHL (SEQ IDNO:381); c) the amino acid sequence of DRVYIHPFHLGGC (SEQ ID NO:382);and d) the amino acid sequence of CDRVYIHPFH (SEQ ID NO:383).

Angiotensin I is cleaved from angiotensinogen (14aa) by thekidney-derived enzyme Renin. Angiotensin I is a biologically inactivepeptide of 10 aa. It is further cleaved at the N-terminus by angiotensinconverting enzyme (ACE) into the biologically active 8aa angiotensin II.This peptide binds to the antgiotensin receptors AT1I and AT2 whichleads to vasoconstriction and aldosterone release.

A vaccine in accordance with the present invention comprising at leastone angiotensin peptide may be used for the treatment of hypertension.

In a particular embodiment of the invention, the antigen or antigenicdeterminant is selected from the group consisting of: (a) a recombinantprotein of HIV, (b) a recombinant protein of Influenza virus (e.g., anInfluenza virus M2 protein or a fragment thereof), (c) a recombinantprotein of Hepatitis C virus, (d) a recombinant protein of Toxoplasma,(e) a recombinant protein of Plasmodium falciparum, (f) a recombinantprotein of Plasmodium vivax, (g) a recombinant protein of Plasmodiumovale, (h) a recombinant protein of Plasmodium malariae, (i) arecombinant protein of breast cancer cells, (j) a recombinant protein ofkidney cancer cells, (k) a recombinant protein of prostate cancer cells,(l) a recombinant protein of skin cancer cells, (m) a recombinantprotein of brain cancer cells, (n) a recombinant protein of leukemiacells, (o) a recombinant profiling, (p) a recombinant protein of beesting allergy, (q) a recombinant proteins of nut allergy, (r) arecombinant proteins of food allergies, (s) recombinant proteins ofasthma, (t) a recombinant protein of Chlamydia, and (u) a fragment ofany of the proteins set out in (a)-(t).

Once the antigen or antigenic determinant of the composition is chosen,at least one second attachment site may be added to the molecule inpreparing to construct the organized and repetitive array associatedwith the non-natural molecular scaffold of the invention. Knowledge ofwhat will constitute an appropriate second attachment site will be knownto those skilled in the art. Representative examples of secondattachment sites include, but are not limited to, the following: anantigen, an antibody or antibody fragment, biotin, avidin, strepavidin,a receptor, a receptor ligand, a ligand, a ligand-binding protein, aninteracting leucine zipper polypeptide, an amino group, a chemical groupreactive to an amino group; a carboxyl group, chemical group reactive toa carboxyl group, a sulfhydryl group, a chemical group reactive to asulfhydryl group, or a combination thereof

The association between the first and second attachment sites will bedetermined by the characteristics of the respective molecules selectedbut will comprise at least one non-peptide bond. Depending upon thecombination of first and second attachment sites, the nature of theassociation may be covalent, ionic, hydrophobic, polar, or a combinationthereof

In one embodiment of the invention, the second attachment site may bethe FOS leucine zipper protein domain or the JUN leucine zipper proteindomain.

In a more specific embodiment of the invention, the second attachmentsite selected is the FOS leucine zipper protein domain, which associatesspecifically with the JUN leucine zipper protein domain of thenon-natural molecular scaffold of the invention. The association of theJUN and FOS leucine zipper protein domains provides a basis for theformation of an organized and repetitive antigen or antigenicdeterminant array on the surface of the scaffold. The FOS leucine zipperprotein domain may be fused in frame to the antigen or antigenicdeterminant of choice at either the amino terminus, carboxyl terminus orinternally located in the protein if desired.

Several FOS fusion constructs are provided for exemplary purposes. Humangrowth hormone (Example 4), bee venom phospholipase A₂ (PLA₂) (Example9), ovalbumin (Example 10) and HIV gp140 (Example 12).

In order to simplify the generation of FOS fusion constructs, severalvectors are disclosed that provide options for antigen or antigenicdeterminant design and construction (see Example 6). The vectors pAV1-4were designed for the expression of FOS fusion in E. coli; the vectorspAV5 and pAV6 were designed for the expression of FOS fusion proteins ineukaryotic cells. Properties of these vectors are briefly described:

1. pAV1:

This vector was designed for the secretion of fusion proteins with FOSat the C-terminus into the E. coli periplasmic space. The gene ofinterest (g.o.i.) may be ligated into the StuI/NotI sites of the vector.

2. pAV2:

This vector was designed for the secretion of fusion proteins with FOSat the N-terminus into the E. coli periplasmic space. The gene ofinterest (g.o.i.) ligated into the NotI/EcoRV (or NotI/HindIII) sites ofthe vector.

3. pAV3:

This vector was designed for the cytoplasmic production of fusionproteins with FOS at the C-terminus in E. coli. The gene of interest(g.o.i.) may be ligated into the EcoRV/NotI sites of the vector.

4. pAV4:

This vector is designed for the cytoplasmic production of fusionproteins with FOS at the N-terminus in E. coli. The gene of interest(g.o.i.) may be ligated into the NotI/EcoRV (or NotI/HindIII) sites ofthe vector. The N-terminal methionine residue is proteolytically removedupon protein synthesis (Hirel et al., Proc. Natl. Acad. Sci. USA86:8247-8251 (1989)).

5. pAV5:

This vector was designed for the eukaryotic production of fusionproteins with FOS at the C-terminus. The gene of interest (g.o.i.) maybe inserted between the sequences coding for the hGH signal sequence andthe FOS domain by ligation into the Eco47III/NotI sites of the vector.Alternatively, a gene containing its own signal sequence may be fused tothe FOS coding region by ligation into the StuI/NotI sites.

6. pAV6:

This vector was designed for the eukaryotic production of fusionproteins with FOS at the N-terminus. The gene of interest (g.o.i.) maybe ligated into the NotI/StuI (or NotI/HindIII) sites of the vector.

As will be understood by those skilled in the art, the construction of aFOS-antigen or -antigenic determinant fusion protein may include theaddition of certain genetic elements to facilitate production of therecombinant protein. Example 4 provides guidance for the addition ofcertain E. coli regulatory elements for translation, and Example 7provides guidance for the addition of a eukaryotic signal sequence.Other genetic elements may be selected, depending on the specific needsof the practitioner.

The invention is also seen to include the production of the FOS-antigenor FOS-antigenic determinant fusion protein either in bacterial (Example5) or eukaryotic cells (Example 8). The choice of which cell type inwhich to express the fusion protein is within the knowledge of theskilled artisan, depending on factors such as whether post-translationalmodifications are an important consideration in the design of thecomposition.

As noted previously, the invention discloses various methods for theconstruction of a FOS-antigen or FOS-antigenic determinant fusionprotein through the use of the pAV vectors. In addition to enablingprokaryotic and eukaryotic expression, these vectors allow thepractitioner to choose between N- and C-terminal addition to the antigenof the FOS leucine zipper protein domain. Specific examples are providedwherein N- and C-terminal FOS fusions are made to PLA₂ (Example 9) andovalbumin (Example 10). Example 11 demonstrates the purification of thePLA₂ and ovalbumin FOS fusion proteins.

In a more specific embodiment, the invention is drawn to an antigen orantigenic determinant encoded by the HIV genome. More specifically, theHIV antigen or antigenic determinant is gp140. As provided for inExamples 11-15, HIV gp140 may be created with a FOS leucine zipperprotein domain and the fusion protein synthesized and purified forattachment to the non-natural molecular scaffold of the invention. Asone skilled in the art would know, other HIV antigens or antigenicdeterminants may be used in the creation of a composition of theinvention.

In another more specific embodiment, the invention is drawn to vaccinecompositions comprising at least one antigen or antigenic determinantencoded by an Influenza viral nucleic acid, and the use of such vaccinecompositions to elicit immune responses. In an even more specificembodiment, the Influenza antigen or antigenic determinant may be an M2protein (e.g., an M2 protein having the amino acids shown in SEQ IDNO:213, GenBank Accession No. P06821, or in SEQ ID NO: 212, PIRAccession No. MFIV62, or fragment thereof (e.g., amino acids from about2 to about 24 in SEQ ID NO:213, the amino acid sequence in SEQ IDNO:212). Further, influenza antigens or antigenic determinants may becoupled to non-natural molecular scaffolds or core particles througheither peptide or non-peptide bonds. When Influenza antigens orantigenic determinants are coupled to non-natural molecular scaffolds orcore particles through peptide bonds, the molecules which form order andrepetitive arrays will generally be prepared as fusion proteinexpression products. The more preferred embodiment is however acomposition, wherein the M2 peptide is coupled by chemicalcross-linking, to Qβ capsid protein HBcAg capsid protein or Piliaccording to the disclosures of the invention.

Portions of an M2 protein (e.g., an M2 protein having the amino acidsequence in SEQ ID NO:213), as well as other proteins against which animmunological response is sought, suitable for use with the inventionmay comprise, or alternatively consist of, peptides of any number ofamino acids in length but will generally be at least 6 amino acids inlength (e.g., peptides 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 97 amino acids inlength).

In another specific embodiment of the invention, the second attachmentsite selected is a cysteine residue, which associates specifically witha lysine residue of the non-natural molecular scaffold or core particleof the invention, or the second attachment site selected is a lysineresidue, which associates specifically with a cysteine residue of thenon-natural molecular scaffold or core particle of the invention. Thechemical linkage of the lysine residue (Lys) and cysteine residue (Cys)provides a basis for the formation of an organized and repetitiveantigen or antigenic determinant array on the surface of the scaffold orcore particle. The cysteine or lysine residue may be engineered in frameto the antigen or antigenic determinant of choice at either the aminoterminus, carboxyl terminus or internally located in the protein ifdesired. By way of example, PLA₂ and HIV gp140 are provided with acysteine residue for linkage to a lysine residue first attachment site.In additional specific embodiments, the invention provides vaccinecompositions suitable for use in methods for preventing and/orattenuating allergic reactions, such as allergic reactions which lead toanaphylaxis. Thus, vaccine compositions of the invention includecompositions which lead to the production of antibodies that preventand/or attenuate allergic reactions. Thus, in certain embodiments,vaccine compositions of the invention include compositions which elicitan immunological response against an allergen. Examples of suchallergens include phospholipases such as the phospholipase A₂ (PLA₂)proteins of Apis mellifera (SEQ ID NO:168, GenBank Accession No. 443189;SEQ ID NO:169, GenBank Accession No. 229378), Apis dorsata (SEQ IDNO:170, GenBank Accession No. B59055), Apis cerana (SEQ ID NO:171,GenBank Accession No. A59055), Bombus pennsylvanicus (SEQ ID NO:172GenBank Accession No. B56338), and Heloderma suspectum (SEQ ID NO:173,GenBank Accession No. P80003; SEQ ID NO:174, GenBank Accession No.514764; SEQ ID NO:175, GenBank Accession No. 226711).

Using the amino acid sequence of a PLA₂ protein of Apis mellifera (SEQID NO:168) for illustration, peptides of at least about 60 amino acidsin length, which represent any portion of the whole PLA₂ sequence, mayalso be used in compositions for preventing and/or attenuating allergicreactions. Examples of such peptides include peptides which compriseamino acids 1-60 in SEQ ID NO:168, amino acids 1-70 in SEQ ID NO:168,amino acids 10-70 in SEQ ID NO:168, amino acids 20-80 in SEQ ID NO:168,amino acids 30-90 in SEQ ID NO:168, amino acids 40-100 in SEQ ID NO:168,amino acids 47-99 in SEQ ID NO:168, amino acids 50-110 in SEQ ID NO:168,amino acids 60-120 in SEQ ID NO:168, amino acids 70-130 in SEQ IDNO:168, or amino acids 90-134 in SEQ ID NO:168, as well correspondingportions of other PLA₂ proteins (e.g., PLA₂ proteins described above).Further examples of such peptides include peptides which comprise aminoacids 1-10 in SEQ ID NO:168, amino acids 5-15 in SEQ ID NO:168, aminoacids 10-20 in SEQ ID NO:168, amino acids 20-30 in SEQ ID NO:168, aminoacids 30-40 in SEQ ID NO:168, amino acids 40-50 in SEQ ID NO:168, aminoacids 50-60 in SEQ ID NO:168, amino acids 60-70 in SEQ ID NO:168, aminoacids 70-80 in SEQ ID NO:168, amino acids 80-90 in SEQ ID NO:168, aminoacids 90-100 in SEQ ID NO:168, amino acids 100-110 in SEQ ID NO:168,amino acids 110-120 in SEQ ID NO:168, or amino acids 120-130 in SEQ IDNO:168, as well corresponding portions of other PLA₂ proteins (e.g.,PLA₂ proteins described above).

Portions of PLA₂, as well as portions of other proteins against which animmunological response is sought, suitable for use with the inventionmay comprise, or alternatively consist of, peptides which are generallyat least 6 amino acids in length (e.g., peptides 6, 7, 8, 9, 10, 12, 15,18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or100 amino acids in length).

PLA₂ peptides (e.g., the full length PLA₂ proteins discussed above, aswell as subportions of each) may also be coupled to any substance (e.g.,a Qβ capsid protein or fragment thereof) which allows for the formationof ordered and repetitive antigen arrays.

In another aspect of the present invention, the invention providescompositions being particularly suitable for treating and/or preventingconditions caused or exacerbated by “self” gene products.

In a preferred embodiment of the invention, the antigenic determinant isRANKL (Receptor activator of NFkB Ligand). RANKL is also known as TRANCE(TNF-related activation induced cytokine), ODF (Osteoclastdifferentiation factor) or OPGL (Osteoprotegerin ligand). The amino acidsequence of the extracellular part of human RANKL is shown in SEQ ID No:221 (RANKL_human: TrEMBL:O14788), while the amino acid sequence of ahuman isoform is shown in SEQ ID No: 222. Sequences for theextracellular part of murine RANKL and an isoform are shown in SEQ IDNo.223 (RANKL_mouse: TrEMBL:O35235), and in SEQ ID No.224 (RANKL_mousesplice forms: TrEMBL:Q9JJK8 and TrEMBL:Q9JJK9), respectively.

It has been shown that RANKL is an essential factor inosteoclastogenesis. Inhibition of the interaction of RANKL with itsreceptor RANK can lead to a suppression of osteoclastogenesis and thusprovide a means to stop excessive bone resorption as seen inosteoporosis and other conditions. The RANKL/RANK interaction wasinhibited either by a RANK-Fc fusion protein or the soluble decoyreceptor of RANKL, termed osteoprotegerin OPG.

In the immune system RANKL is expressed on T cells while RANK is foundon antigen-presenting cells. The RANKL-RANK interaction was shown to becritical for CD40L-independent T-helper cell activation (Bachmann etal., J. Exp. Med. 7: 1025 (1999)) and enhance the longevity and adjuvantproperties of dendritic cells (Josien et al., J Exp Med. 191: 495(2000)).

In bone RANKL is expressed on stromal cells or osteoblasts, while RANKis expressed on the osteoclast precursor. The interaction of RANK andRANKL is crucial for the development of osteoclast precursors to matureosteoclasts. The interaction can be blocked by osteoprotegerin.

OPG-deficient mice develop osteoporosis that can be rescued by injectionof recombinant OPG. This means that OPG is able to reverse osteoporosis.Thus, inhibition of the RANK-RANKL interaction by way of injecting thisspecific embodiment of the invention may reverse osteoporosis.

In addition, arterial calcification was observed in OPG k.o. mice whichcould be reversed by injection of OPG (Min et al., J. Exp. Med. 4: 463(2000)). In an adjuvant-induced arthritis model OPG injection was ableto prevent bone loss and cartilage destruction, but not inflammation(paw swelling). It is assumed that activated T cells lead to aRANKL-mediated increase of osteoclastogenesis and bone loss. OPGinhibits prostate cancer-induced osteoclastogenesis and preventsprostate tumor growth in the bone of mice. OPG diminishes advanced bonecancer pain in mice.

RANKL is a transmembrane protein of 245 aa belonging to theTNF-superfamily. Part of the extracellular region (178 aa) can be shedby a TACE-like protease (Lum et al., J Biol Chem. 274:13613 (1999)). Inaddition splice variants lacking the transmembrane domain have beendescribed (Ikeda et al., Endocrinology 142: 1419 (2001)). The shed partcontains the domain highly homologous to soluble TNF-α. Thisextracellular domain of RANKL forms homotrimers as seen for TNF-α. TheC-terminus seems to be involved in the trimer contact site. One cysteineis present in this region of the sequence.

We have built a model for the 3-dimensional structure of thecorresponding region of RANKL and found that the naturally presentcysteine may not be accessible in the folded structure for interactionwith a first attachment site on the carrier in accordance with thepresent invention. The N-terminus is preferred for attaching a secondattachment site comprising an amino acid linker with an additionalcysteine residue. A human-RANKL construct with an N terminal amino acidlinker containing a cysteine residue fused to the extracellular part ofRANKL is a very preferred embodiment of the invention. However, anamino-acid linker containing a cysteine residue as second attachmentsite and being fused at the C-terminus of the RANKL sequence or theextracellular part of RANKL leads to further preferred embodiments ofthe invention.

Human-RANKL constructs, such as the one identified in SEQ ID NO:320, aregenerated according to the teachings disclosed in EXAMPLE 6, and the manskilled in the art are able to compare murine and human RANKL sequencesin a protein sequence alignment to identify the part of the sequence ofhuman-RANKL to be cloned in the vectors described in EXAMPLE 6.Fragments containing amino acids 138-317 and corresponding to theC-terminal region of the extracellular domain of human RANKL, areparticularly favored embodiments of the invention, and can be modifiedfor coupling to VLPs and Pili as required according to the teaching ofthe present invention. However, other suitable vectors may also be usedfor expression in the suitable host described below. Further human-RANKLconstructs, and in particular, the ones comprising the part of theextracellular region (178 aa), —or fragments thereof—that can be shed bya TACE-like protease (Lum et al., J Biol Chem. 274:13613 (1999)), orcomprising the sequence corresponding to the alternative splice variantslacking the transmembrane domain, as well as conservative fragmentsthereof, are intended to be encompassed within the scope of the presentinvention. Human C-terminal fragments comprising amino acids 165-317 arealso embodiments of the invention. Alternatively, fragments whichencompass the entire extracellular region (amino acids 71-317) and canbe modified for coupling to VLPs and Pili as required according to theteaching of the present invention, are also within the scope of theinvention.

RANKL has been expressed in different systems (E. coli, insect cells,mammalian cells) and shown to be active, and therefore severalexpression systems can be used for production of the antigen of thecomposition. In the case where expression of the protein is directed tothe periplasm of E. coli, the signal peptide of RANKL, or of RANKLconstructs consisting of the extracellular part of the protein, and bothpossibly modified to comprise a second attachment site in accordancewith the invention, is replaced with a bacterial signal peptide. Forexpression of the protein in the cytoplasm of E. coli, RANKL constructsare devoid of signal peptide.

In another preferred embodiment of the invention, the antigenicdeterminant is MIF or a fragment thereof. MIF is a cytokine that hasbeen first described in 1966 by its function as an inhibitor ofmacrophage migration. It is also known as delayed early response protein6 (DER6), glycosylation inhibiting factor or phenylpyruvate tautomerase.The latter name originates from enzymatic activity of MIF, however theendogenous substrate has not been identified.

MIF has been shown to be implicated in a wide range of conditions. MIF(mRNA and protein) is upregulated in delayed type hypersensitivity (DTH)reaction induced by tuberculin, and anti-MIF antibody inhibits this DTHreaction. MIF is also upregulated in renal allograft rejection. In amodel for ocular autoimmune disease, experimental autoimmuneuveoretinitis (EAU), anti-MIF treatment caused delay of EAU development.In patients, there is an increase in serum of MIF, which is also thecase in Behcet's disease patients and patients suffering fromiridocyclitis. Immunization against MIF may provide a way of treatmentagainst rheumatoid arthritis.

High serum MIF concentration has been found in atopic dermatitispatients. In skin lesions, MIF is diffusely expressed instead of beingfound in the basal cell layer in controls. MIF concentration isdecreasing after steroid treatment, consistent with a role of MIF ininflammation. MIF has also been found to contribute to the establishmentof glomerulonephritis. Animals treated with anti-MIF Antibody showsignificantly reduced glomerulonephritis. MIF is pituitary derived,secreted e.g. upon LPS stimulation, and potentiates endotoxemia.Accordingly, anti-MIF mAb inhibits endotoxemia and septic shock, whilerecombinant MIF markedly increases lethality of peritonitis. MIF is alsoa glucocorticoid-induced modulator of cytokine production, and promotesinflammation.

MIF is produced by T-cells (Th2), supports proliferation of T-cells, andanti-MIF-treatment reduces T-cell proliferation and IgG levels. There isan increased MIF concentration in the cerebrospinal fluid of multiplesclerosis and neuro-Behcet's disease patients. High MIF levels were alsofound in sera of patients with extended psoriasis. High MIF levels arefound in sera of ulcerative colitis patients but not Crohn's diseasepatients.

High MIF levels have been found in sera of patients with bronchicasthma. MIF is also upregulated in synovial fluid of rheumatoidarthritis patients. Anti-MIF treatment was effectively decreasingrheumatoid arthritis in mouse and rat models (Mikulowska et al., J.Immunol. 158:5514-7(1997); Leech et al., Arthritis Rheum. 41:910-7(1998), Leech et al. Arthritis Rheum. 43:827-33 (2000), Santos et al.,Clin. Exp. Immunol. 123:309-14 (2001)). Thus, treatment directed atinhibiting MIF activity using a composition comprising MIF as anantigenic determinant may be beneficial for the conditions mentionedabove.

MIF from mouse, rat and human consists of 114 amino acid and containsthree conserved cysteines, as shown in SEQ ID No 225 (MIF_rat:SwissProt), in SEQ ID No 226 (MIF_mouse: SwissProt) and in SEQ ID No 227(MIF_human: SwissProt). Three subunits form a homotrimer that is notstabilized by disulfide bonds. The X-ray structure has been solved andshows three free cysteines (Sun et al., PNAS 93: 5191-96 (1996)), whilesome literature data claim the presence of a disulfide bond.Nonetheless, none of the cysteines are exposed enough for optimalinteraction with a possible first attachment site present on thecarrier. Thus, as the C-terminus of the protein is exposed in the trimerstructure, an amino acid linker containing a free cysteine residue is,preferably, added at the C-terminus of the protein, for generation ofthe second attachment site in this preferred embodiment of theinvention, as exemplarily described in EXAMPLE 4 for rat-MIF.

There is only one amino acid change between mouse- and rat-MIF, andsimilarly a very high sequence homology (about 90% sequence identity)between human- and rat-MIF or human- and mouse-MIF. Human- and mouse-MIFconstructs according to the invention are described and can be generatedas disclosed in EXAMPLE 4. In order to demonstrate the high potency toinduce a self-specific immune response of MIF protein, or fragmentsthereof, associated to a core particle in accordance with the presentinvention, rat-MIF constructs coupled to Qβ capsid protein were injectedin mice. The high antibody titers obtained by immunizing mice withrat-MIF show that tolerance towards immunization with self-antigens wasovercome by immunizing with MIF constructs coupled to virus-likeparticles, and in particular to Qβ capsid protein (EXAMPLE 4).Therefore, compositions in accordance with the present inventioncomprising human-MIF protein associated to a core particle, preferablyto pili or a virus-like particle, and more preferably to a virus-likeparticle of a RNA-phage, and even more preferably to RNA-phage Qβ or fr,represent very preferred embodiments of the present invention.

However, an amino acid linker containing a free cysteine that is addedat the N-terminus of the sequence of MIF leads to further preferredembodiments of the present invention. MIF has been expressed in E. coli,purified and shown to be fully functional (Bernhagen et al.,Biochemistry 33: 14144-155 (1994). Thus, MIF may be, preferably,expressed in E. coli for generating the preferred embodiments of theinvention.

Tautomerase activity of MIF is inhibited, if the start methionine is notcleaved from the construct. MIF constructs expressed in E. coli anddescribed in EXAMPLE 4 show tautomerase activity. Mutants of MIF wherethe start methionine is cleaved and where the proline residue rightafter the start methionine in the sequence is mutated to alanine also donot show tautomerase activity represent further embodiments of theinvention and are intended to be encompassed within the scope of theinvention. In some specific embodiments, immunization with MIF mutantsdevoid of tautomerase activity is envisaged.

In another preferred embodiment of the invention, the antigenicdeterminant is Interleukin-17 (IL-17). Human IL-17 is a 32-kDa,disulfide-linked, homodimeric protein with variable glycosylation (Yao,Z. et al., J. Immunol. 155: 5483-5486 (1995); Fossiez, F. et al., J.Exp. Med. 183: 2593-2603 (1996)). The protein comprises 155 amino acidsand includes an N-terminal secretion signal sequence of 19-23 residues.The amino acid sequence of IL-17 is similar only to a Herpesvirusprotein (HSV13) and is not similar to other cytokines or known proteins.The amino acid sequence of human IL-17 is shown in SEQ ID No: 228(ACCESSION #: AAC50341), The mouse protein sequence is shown in SEQ IDNo: 229 (ACCESSION #: AAA37490). Of the large number of tissues and celllines evaluated, the mRNA transcript encoding IL-17 has been detectedonly in activated T cells and phorbol 12-myristate13-acetate/ionomycin-stimulated peripheral blood mononuclear cells (Yao,Z. et al., J. Immunol. 155: 5483-5486 (1995); Fossiez, F. et al., J.Exp. Med. 183: 2593-2603 (1996)). Both human and mouse sequences contain6 cysteine residues.

The receptor for IL-17 is widely expressed in many tissues and celltypes (Yao, Z. et al., Cytokine 9: 794-800 (1997)). Although the aminoacid sequence of the human IL-17 receptor (866 aa) predicts a proteinwith a single trans-membrane domain and a long, 525 aa intracellulardomain, the receptor sequence is unique and is not similar to that ofany of the receptors from the cytokine/growth factor receptor family.This coupled with the lack of similarity of IL-17 itself to other knownproteins indicates that IL-17 and its receptor may be part of a novelfamily of signalling protein and receptors. Clinical studies indicateIL-17 may be involved in many inflammatory diseases. IL-17 is secretedby synovial T cells from rheumatoid arthritis patients and stimulatesthe production of inflammatory mediators (Chabaud, M. et al., J.Immunol. 161: 409-414 (1998); Chabaud, M. et al., Arthritis Rheum. 42:963-970 (1999)). High levels of IL-17 have been reported in patientswith rheumatoid arthritis (Ziolkowska M. et al., J Immunol. 164:2832-8(2000)).

Interleukin 17 has been shown to have an effect on proteoglycandegradation in murine knee joints (Dudler J. et al., Ann Rheum Dis. 59:529-32 (2000)) and contribute to destruction of the synovium matrix(Chabaud M. et al., Cytokine. 12:1092-9 (2000)). There are relevantarthritis models in animals for testing the effect of an immunizationagainst MIF (Chabaud M. et al., Cytokine. 12:1092-9 (2000)). Elevatedlevels of IL-17 mRNA have been found in mononuclear cells from patientswith multiple sclerosis (Matusevicius, D. et al., Mult. Scler. 5:101-104 (1999)). Elevated serum levels of IL-17 are observed in patientssuffering Systemic Lupus Erythematosus (Wong C. K. et al., Lupus 9:589-93 (2000)). In addition, IL-17 mRNA levels are increased in T cellsisolated from lesional psoriatic skin (Teunissen, M. B. et al., J.Invest. Dermatol. 111: 645-649 (1998)).

The involvement of IL-17 in rejection of kidney graft has also beendemonstrated (Fossiez F. et al., Int. Rev. Immunol. 16:541-51 (1998)).Evidence for a role of IL-17 in organ allograft rejection has also beenpresented by Antonysamy et al. (J. Immunol. 162:577-84 (1999)) whoshowed IL-17 promotes the functional differentiation of dendritic cellprogenitors. Their findings suggest a role for IL-17 in allogeneic Tcell proliferation that may be mediated in part via amaturation-inducing effect on DCs. Furthermore the same group reports(Tang J. L. et al., Transplantation 72:348-50 (2001)) a role for IL-17in the immunopathogenesis of acute vascular rejection whereInterleukin-17 antagonism inhibits acute but not chronic vascularrejection. IL-17 appears to have potential as a novel target fortherapeutic intervention in allograft rejection.

The above findings suggest IL-17 may play a pivotal role in theinitiation or maintenance of an inflammatory response (Jovanovic, D. V.et al., J. Immunol. 160: 3513-3521 (1998)).

The anti-IL-17 monoclonal antibody mAb5 (Schering-Plough ResearchInstitute) was able to completely inhibit the production of IL-6 fromrheumatoid arthritis (RA) synovium supernatants following induction by50 ng/ml of IL-17. An irrelevant mAb MX1 had no effect in this assay.mAb5 is a mouse IgG1 obtained after immunization with human rIL-17(r=recombinant). A concentration of 1 μg/ml of mAb5 was able tocompletely inhibit the IL-6 production in the assay system (Chabaud, M.et al., J. Immunol. 161: 409-414 (1998)). Thus, immunization againstIL-17 provides a way of treatment for the various conditions describedabove.

In another preferred embodiment of the invention, thus, the compositioncomprises a linker containing a second attachment site and being fusedto the C-terminus of recombinant IL-17. In further preferred embodimentsof the invention, however, an amino acid linker containing a freecysteine is fused to the N-terminus of the sequence corresponding to thesequence of the processed protein, or inserted at the N-terminus of thesequence of the mature form of the protein, C-terminally of the signalpeptide. For eukaryotic expression systems, the signal peptide of theIL-17 gene, as it is the case for the other self-antigens indicatedherein, may be replaced by another signal peptide if required. Forexpression in bacteria, the signal peptide is either replaced by abacterial signal peptide for soluble expression in the periplasm, ordeleted for expression in the cytoplasm. Constructs of human IL-17devoid of signal peptide will preferably comprise residues 24-155,22-155, 21-155 or 20-155. Constructs of mouse IL-17 devoid of signalpeptide will preferably comprise residues 26-158, 25-158, 24-158 or27-155. Human IL-17 may be expressed in CV1/EBNA cells; recombinanthIL-17 has been shown to be secreted in both glycosylated andnonglycosylated forms (Yao, Z. et al., J. Immunol. 155: 5483-5486(1995)). IL-17 can also be expressed as hIL-17/Fc fusion protein, withsubsequent cleavage of the IL-17 protein from the fusion protein. IL-17may also be expressed in the yeast Pichia pastoris (Murphy K. P. et.al., Protein Expr Purif. 12: 208-14 (1998)). Human IL-17 may also beexpressed in E. coli. When expression of IL-17 in E. coli is directed tothe periplasm, the signal peptide of IL-17 is replaced by a bacterialsignal peptide. For expression of the protein in the cytoplasm of E.coli, IL-17 constructs are devoid of signal peptide.

In another preferred embodiment of the invention the antigenicdeterminant is Interleukin-13 (IL-13). IL-13 is a cytokine that issecreted by activated T lymphocytes and primarily impacts monocytes,macrophages, and B cells. The amino acid sequence of precursor humanIL-13 is shown in SEQ ID No: 230 and the amino acid sequence ofprocessed human IL-13 is shown in SEQ ID No: 231. The first 20 aminoacids of the precursor protein correspond to the signal peptide, and areabsent of the processed protein. The mouse sequence has also beendescribed, and the processed amino acid sequence is shown in SEQ ID No:232 (Brown K. D. et al., J Immunol. 142:679-687 (1989)). Depending onthe expression host, the IL-13 construct will comprise the sequence ofthe precursor protein, e.g. for expression and secretion in eukaryotichosts, or consist of the mature protein, e.g. for cytoplasmic expressionin E. coli. For expression in the periplasm of E. coli, the signalpeptide of IL-13 is replaced by a bacterial signal peptide.

IL-13 is a T helper 2-derived cytokine (like IL-4, IL-5) that hasrecently been implicated in allergic airway responses (asthma).Upregulation of IL-13 and IL-13 receptor has been found in many tumourtypes (e.g. Hodgkin lymphoma). Interleukin 13 is secreted by andstimulates the growth of Hodgkin and Reed-Sternberg cells (Kapp U etal., J Exp Med. 189:1939-46 (1999)). Thus, immunization against IL-13provides a way of treating among others the conditions described above,such as Asthma or Hodgkins Lymphoma.

Preferably, the composition comprises an amino acid linker containing afree cysteine residue and being fused to the N or C-terminus of thesequence of mature IL-13 to introduce a second attachment site withinthe protein. In further preferred embodiments, an amino acid linkercontaining a free cysteine is added to the N-terminus of the mature formof IL-13, since it is freely accessible according to the NMR structureof IL-13 (Eisenmesser, E. Z. et al., J. Mol. Biol. 310: 231 (2001)). Inagain further preferred embodiments, the amino acid linker containing afree cysteine is fused to the N-terminus of the sequence correspondingto the sequence of the processed protein, or inserted at the N-terminusof the sequence of the mature form of the protein, C-terminally of thesignal peptide. In still further preferred embodiments, an amino acidlinker containing a free cysteine residue is added to the C-terminus ofthe protein.

IL-13 may be expressed in E. coli (Eisenmesser E. Z. et al., ProteinExpr. Purif. 20:186-95 (2000)), or in NS-0 cells (eukaryotic cell line)(Cannon-Carlson S. et al., Protein Expr. Purif. 12:239-48 (1998)).EXAMPLE 9 describes constructs and expression of constructs of murineIL-13, fused to an amino acid linker containing a cysteine residue, inbacterial and eukaryotic hosts. Human IL-13 constructs can be generatedaccording to the teachings of EXAMPLE 9 and yielding the proteins humanC-IL-13-F (SEQ ID NO:330) and human C-IL-13-S(SEQ ID NO:331) afterexpression of the fusion proteins and cleavage with Factor Xa, andenterokinase respectively. The so generated proteins can be coupled toVLPs and Pili, leading to preferred embodiments of the invention.

In yet another embodiment of the invention, the antigenic determinant isInterleukin-5 (IL-5). IL-5 is a lineage-specific cytokine foreosinophilopoiesis and plays an important part in diseases associatedwith increased number of eosinophils, such as asthma. The sequence ofprecursor and processed human IL-5 is provided in SEQ ID No: 233 and inSEQ ID No: 234, respectively, and the processed mouse amino acidsequence is shown in SEQ ID No: 235.

The biological function of IL-5 has been shown in several studies(Coffman R. L. et al., Science 245: 308-10 (1989); Kopf et al., Immunity4:15-24 (1996)), which point to a beneficial effect of inhibiting IL-5function in diseases mediated through eosinophils Inhibition of theaction of IL-5 provides thus a way of treatment against asthma and otherdiseases associated with eosinophils.

IL-5 forms a dimer, covalently linked by a disulfide bridge. A singlechain (sc) construct has been reported wherein two monomers of IL-5 arelinked by a peptide linker.

In preferred embodiments of the invention, a peptide linker containing afree cysteine is added at the N-terminus of the sequence of theprocessed form of IL-5. Addition of a linker containing a free cysteineis also, preferably, envisaged at the N-terminus of the sequence of theprocessed form of a scIL-5. In further preferred embodiments, the aminoacid linker containing a free cysteine is fused to the N-terminus of thesequence corresponding to the sequence of the processed protein, orinserted at the N-terminus of the sequence of the mature form of theprotein, C-terminally of the signal peptide.

In again further preferred embodiments, a linker containing a freecysteine is fused to the C-terminus of the sequence of IL-5, or to theC-terminus of a scIL-5 sequence.

A number of expression systems have been described for IL-5 and can beused in preparing the compositions of the invention. A bacterialexpression system using E. coli has been described by Proudfoot et al.,(Biochem J. 270:357-61 (1990)). In the case where IL-5 is expressed inthe cytoplasm of E. coli, the IL-5 construct is devoid of a signalpeptide. Insect cells may also be used for producing IL-5 constructs formaking the compositions of the invention (Pierrot C. et al., Biochem.Biophys. Res. Commun. 253:756-60 (1998)). Likewise, Baculovirusexpression systems (sf9 cells; Ingley E. et al., Eur. J. Biochem.196:623-9 (1991) and Brown P.M. et al., Protein Expr. Purif. 6: 63-71(1995)) can also be used. Finally, mammalian expression systems havealso been reported (CHO cells) and can be used in preparing thesecompositions of the invention (Kodama S et al., J. Biochem. (Tokyo)110:693-701 (1991)).

Baculovirus expression systems (Mitchell et al., Biochem. Soc. Trans.21:332S (1993); Kunimoto D Y et al., Cytokine 3:224-30 (1991)) and amammalian cell expression system using CHO cells (Kodama S et al.,Glycobiology 2:419-27 (1992)) have also been described for mouse IL-5.

EXAMPLE 10 describes the expression of murine IL-5 constructs whereinthe IL-5 sequence is fused at its N-terminus to amino acid linkerscontaining a cysteine residue for coupling to VLPs and Pili. Humanconstructs can be generated according to the teaching of EXAMPLE 10 andyield the proteins human C-IL-5-E (SEQ ID NO:335), human C-IL-5-F (SEQID NO:336) and human C-IL-5-S: (SEQ ID NO:337) suitable for coupling toVLPs and Pili and leading to preferred embodiments of the invention.

In another preferred embodiment of the invention, the antigenicdeterminant is CCL-21. CCL-21 is a chemokine of the CC subfamily that isalso known as small inducable cytokine A21, as exodus-2, as SLD(secondary lymphocyte cytokine), as TCA4 (thymus-derived chemotacticagent 4) or 6Ckine.

CCL21 inhibitis hemopoiesis and stimulates chemotaxis for thymocytes,activated T-cells and dendritic cells, but not for B cells, macrophagesor neutrophiles. It shows preferential activity towards naive T cells.It is also a potent mesangial cell chemoattractant. CCL21 binds tochemokine receptors CCR7 and to CXCR3 (dependent on species). It cantrigger rapid integrin-dependent arrest of lymphocytes rolling underphysiological shear and is highly expressed by high endothelial venules.

Murine CCL21 inhibited tumor growth and angiogenesis in a human lungcancer SCID mouse model (Arenberg et al., Cancer Immunol. Immunother.49: 587-92 (2001)) and a colon carcinoma tumor model in mice (Vicari etal., J. Immunol. 165: 1992-2000 (2001)). The angiostatic activity ofmurine CCL21 was also detected in a rat corneal micropocket assay (Sotoet al., Proc. Natl. Acad. Sci. USA 95: 8205-10 (1998).

It has been shown that chemokine receptors CCR7 and CXCR4 areupregulated in breast cancer cells and that CCL21 and CXCL12, therespective ligands, are highly expressed in organs representing thefirst destinations of breast cancer metastasis Müller et al. (Nature410: 50-6 (2001)). In vitro CCL21-mediated chemotaxis could be blockedby neutralizing anti-CCL21 antibodies as was CXCR4-mediated chemotaxisby the respective antibodies. Thus, immunization against CCL21 providesa way of treatment against metastasis spread in cancers, morespecifically in breast cancer.

Secreted CCL21 consist of 110 or 111 aa in mice and humans,respectively. The respective sequences are shown in SEQ ID No: 236(Swissprot: SY21_human) and in SEQ ID No: 237 (Swissprot: SY21_mouse).In contrast to other CC cytokines does CCL21 contain two more cysteineswithin an extended region at the C-terminus. It is assumed that allcysteines are engaged in disulfide bonds.

In the following, constructs and expression systems are described formaking compositions of the invention comprising the CCL21 antigenicdeterminant. In the NMR structure of the homologous protein eotaxin,both N- and C-terminus are exposed to the solvent. In some specificembodiments, an amino acid linker containing a free cysteine residue asa second attachment site is added at the C-terminus of the protein. Afusion protein with alkaline phosphatase (at the C-terminus of CCL21)has been expressed and was shown to be functional, showing that fusionsat the C-terminus of CCL21 are compatible with receptor binding. Inother specific embodiments, the amino acid linker containing a freecysteine is fused to the N-terminus of the sequence corresponding to thesequence of the processed protein, or inserted at the N-terminus of thesequence of the mature form of the protein, C-terminally of the signalpeptide.

Several expression systems have been described for production of CCL21(e.g. Hedrick et al., J Immunol. 159: 1589-93 (1997)). For example, itmay expressed in a baculovirus system (Nagira et al., J. Biol. Chem.272: 19518-24 (1997)).

In a related preferred embodiment, the antigenic determinant is Stromalderived factor-1 (SDF-1), now termed CXCL12. CXCL12 is a chemokineproduced by bone marrow stromal cells and was originally identified as astimulatory factor for pre-B cells.

As already stated above, it has been shown that chemokine receptors CCR7and CXCR4 are upregulated in breast cancer cells and that CCL21 andSDF-1, the respective ligands, are highly expressed in organsrepresenting the first destinations of breast cancer metastasis Mülleret al. (Nature 410: 50-6 (2001)). In vitro SDF-1/CXCR4-mediatedchemotaxis could be inhibited by neutralizing anti-SDF-1 and anti-CXCR4antibodies.

In a breast cancer metastasis model in SCID mice using the humanMDA-MB-231 breast cancer cell line, a significant decrease in lungmetastasis was observed when mice were treated with anti-CXCR4antibodies. In the draining lymph nodes a reduction of metastasis to theinguinal and axillary lymph nodes (38% instead of 100% metastasis incontrols) was observed. Thus, immunization against CXCL12 provides a wayof treatment against metastasis of cancers, more specifically of breastcancers.

The SDF-1/CXCR4 chemokine-receptor pair has been shown to increase theefficacy of homing of more primitive hematopoietic progenitor cells tobe bone marrow. In addition, CXCR4 and SDF-1 are supposed to influencethe distribution of chronic lymphocytic leukemia cells. These cellsinvariably infiltrate the bone marrow of patients and it was shown thattheir migration in the bone marrow was CXCR4 dependent. Chroniclymphocytic leukemia cells undergo apoptosis unless they are coculturedwith stromal cells. SDF-1 blocking antibodies could inhibit thisprotective effect of stromal cells (Burger et al., Blood 96: 2655-63(2000)) Immunizing against CXCL12 thus provides a way of treatmentagainst chronic lymphocytic leukemia.

CXCR4 has been shown to be a coreceptor for entry of HIV into T-cells.SDF-1 inhibits infection of CD4+ cells by X4 (CXCR4-dependent) HIVstrains (Oberlin et al., Nature 382:833-5 (1996); Bleul et al., Nature382:829-33 (1996), Rusconi et al., Antivir. Ther. 5:199-204 (2000)).Synthetic peptide analogs of SDF-1 have been shown to effectivelyinhibit HIV-1 entry and infection via the CXCR4 receptor (WO059928A1).Thus, immunization against CXCL12 provides a way to block HIV entry inT-cells, and therefore a way of treating AIDS.

SDF-1-CXCR4 interactions were also reported to play a central role inCD4+ T cell accumulation in rheumatoid arthritis synovium (Nanki et al.,2000). Immunization against SDF-1 thus provides a way of treatmentagainst rheumatoid arthritis.

Human and murine SDF-1 are known to arise in two forms, SDF-1α andSDF-1β, by differential splicing from a single gene. They differ in fourC-terminal amino acids that are present in SDF-1β (74 aa) and absent inSDF-1α (70 aa). The sequence of human is shown in SEQ ID No: 238(Swissprot: SDF1_human) and the sequence mouse SDF-1 is shown in SEQ IDNo: 239 (Swissprot: SDF1_mouse). SDF-1 contains four conserved cysteinesthat form two intra-molecular disulfide bonds. The crystal structure ofSDF shows a non covalently-linked dimer (Dealwis et al., PNAS 95:6941-46 (1998)). The SDF-1 structure also shows a long N-terminalextension.

Alanine-scanning mutagenesis was used to identify (part of) thereceptor-binding site on SDF-1 (Ohnishi et al., J. Interferon CytokineRes. 20: 691-700 (2000)) and Elisseeva et al. (J. Biol. Chem.275:26799-805 (2000)) and Heveker et al. (Curr. Biol. 8:369-76 (1998))described SDF-1 derived peptides inhibiting receptor binding (and HIVentry).

In the following, constructs and expression systems suitable in thegeneration of the compositions of the invention related to SDF-1 aredescribed. The N- and C-terminus of SDF-1 are exposed to the solvent. Inspecific embodiments, an amino acid linker containing a cysteine assecond attachment site is thus fused to the C-terminus of the proteinsequence, while in other specific embodiments an amino acid linkercontaining a cysteine as second attachment site is fused to theN-terminus of the protein sequence. The amino acid linker containing afree cysteine is fused to the N-terminus of the sequence correspondingto the sequence of the processed protein, or inserted at the N-terminusof the sequence of the mature form of the protein, C-terminally of thesignal peptide. The genes coding for these specific constructs may becloned in a suitable expression vector.

Expression of SDF-1 in a sendai virus system in chicken embryonicfibroblasts (Moriya et al., FEBS Lett. 425:105-11 (1998)) has beendescribed as well as expression in E. coli (Holmes et al., Prot. Expr.Purif. 21: 367-77 (2001)) and chemical synthesis of SDF-1 (Dealwis etal., PNAS 95: 6941-46 (2001)).

In yet another embodiment of the invention, the antigenic determinant isBLC. B-lymphocyte chemoattractant (BLC, CXCL13) is expressed in thespleen, Peyer's patches and lymph nodes (Gunn et al., 1998). Itsexpression is strongest in the germinal centres, where B cells undergosomatic mutation and affinity maturation. It belongs to the CXCchemokine family, and its closest homolog is GROα_ (Gunn et al., Nature391:799-803 (1998)). Human BLC is 64% homologous to murine BLC. Itsreceptor is CXCR5. BLC also shares homology with IL-8. BLC recruitsB-cells to follicles in secondary lymphoid organs such as the spleen andpeyer's patches. BLC is also required for recruitment of B-cells tocompartment of the lymph nodes rich in follicular Dendritic Cells (FDCs)(Ansel et al., Nature 406:309-314 (2000)). BLC also induces increasedexpression of Lymphotoxinα1β2 (LT?α1β2) on the recruited B-cells. Thisprovides a positive feed-back loop, since LT?α1β2 promotes BLCexpression (Ansel et al., Nature 406:309-314 (2000)). BLC has also beenshown to be able to induce lymphoid neogenesis (Luther et al., Immunity12:471-481 (2000)). It appears that FDCs also express BLC. Thusimmunization against BLC may provide a way of treatment againstautoimmune diseases where lymphoid neogenesis is involved, such asRheumatoid synovitis and Rheumatoid arthritis or Type I diabetes. Aconstruct of BLC bearing a C-terminal his-tag has been described, and isfunctional (Ansel, K. M. et al., J. Exp. Med. 190: 1123-1134 (1999)).

Thus, in a preferred embodiment of the present invention, thecomposition comprises a linker containing a cysteine residue as secondattachment site and being fused at the C-terminus of the BLC sequence.

In IL-8, which is homologous to BLC, both N- and C-termini are free. Ina further preferred embodiment, addition of an amino acid linkercontaining a cysteine residue as second attachment site is, therefore,done to the N-terminus of BLC for generation of this specificcomposition of the invention.

In further preferred embodiments of the present invention, thecomposition comprises an amino acid linker containing a free cysteineand being fused to the N-terminus of the sequence corresponding to thesequence of the processed protein, or inserted at the N-terminus of thesequence of the mature form of the protein, C-terminally of the signalpeptide. The genes coding for these specific constructs may be cloned ina suitable expression vector and expressed accordingly. The sequence ofhuman BLC is shown in SEQ ID No: 240 (Accession: NP_(—)006410). Aminoacids 1-22 of the sequence are the signal peptide. The mouse sequence isshown in SEQ ID No: 241 (Accession NP_(—)061354). Amino acids 1-21 arethe signal peptide. Compositions of the invention with BLC as theantigenic determinant, preferably, use the mature form of the proteinfor generating the compositions of the invention.

In another specific embodiment, the antigenic determinant is Eotaxin.Eotaxin is a chemokine specific for Chemokine receptor 3, present oneosinophils, basophils and Th2 cells. Eotaxin seems however to be highlyspecific for Eosinophils (Zimmerman et al., J. Immunol. 165: 5839-46(2000)). Eosinophil migration is reduced by 70% in the eotaxin-1knock-out mouse, which however can still develop eosinophilia(Rothenberg et al., J. Exp. Med. 185: 785-90 (1997)). IL-5 seems to beresponsible for the migration of eosinophils from bone-marrow to blood,and eotaxin for the local migration in the tissue (Humbles et al., J.Exp. Med. 186: 601-12 (1997)).

The human genome contains 3 eotaxin genes, eotaxin1-3. They share 30%homology to each other. Two genes are known so far in the mouse: eotaxin1 and eotaxin 2 (Zimmerman et al., J. Immunol. 165: 5839-46 (2000)).They share 38% homology. Murine eotaxin-2 shares 59% homology with humaneotaxin-2. In the mouse, eotaxin-1 seems to be ubiquitously expressed inthe gastro-intestinal tract, while eotaxin-2 seems to be predominantlyexpressed in the jejunum (Zimmerman et al., J. Immunol. 165: 5839-46(2000)). Eotaxin-1 is present in broncho-alveolar fluid (Teixeira etal., J. Clin. Invest. 100: 1657-66 (1997)). The sequence of humaneotaxin-1 is shown in SEQ ID No.: 242 (aa 1-23 corresponds to the signalpeptide), the sequence of human eotaxin-2 is shown in SEQ ID No.: 243(aa 1-26 corresponds to the signal peptide), the sequence of humaneotaxin-3 is shown in SEQ ID No.: 244 (aa 1-23 corresponds to the signalpeptide), the sequence of mouse eotaxin-1 is shown in SEQ ID No.: 245(aa 1-23 corresponds to the signal peptide), and the sequence of mouseeotaxin-2 is shown in SEQ ID No.: 246 (aa 1-23 corresponds to the signalpeptide).

Eotaxin has a MW of 8.3 kDa. It is in equilibrium between monomers anddimers over a wide range of conditions, with an estimated Kd of 1.3 mMat 37° C. (Crump et al., J. Biol. Chem. 273: 22471-9 (1998)). Themonomer form is however predominant. The structure of Eotaxin has beenelucidated by NMR spectroscopy. Binding site to its receptor CCR3 is atthe N-terminus, and the region preceding the first cysteine is crucial(Crump et al., J. Biol. Chem. 273: 22471-9 (1998)). Peptides ofchemokine receptors bound to Eotaxin confirmed this finding. Eotaxin hasfour cysteines forming two disulfide bridges. Therefore, in a preferredembodiment, the inventive composition comprises an amino-acid linkercontaining a cysteine residue as second attachment site and being,preferably, fused to the C-terminus of the Eotaxin sequence. In otherpreferred embodiments, an amino acid linker containing a free cysteineis fused to the N-terminus of the sequence corresponding to the sequenceof the processed protein, or inserted at the N-terminus of the sequenceof the mature form of the protein, C-terminally of the signal peptide.The genes coding for these specific constructs are cloned in a suitableexpression vector.

Eotaxin can be chemically synthesized (Clark-Lewis et al., Biochemistry30:3128-3135 (1991)). Expression in E. coli has also been described forEotaxin-1, in the cytoplasm (Crump et al., J. Biol. Chem. 273: 22471-9(1998)). Expression in E. coli as inclusion bodies with subsequentrefolding (Mayer et al., Biochemistry 39: 8382-95 (2000)), and Insectcell expression (Forssmann et al., J. Exp. Med. 185: 2171-6 (1997)) havebeen described for Eotaxin-2, and may, moreover, be used to arrive atthe specific embodiments of the invention.

In yet another specific embodiment of the invention, the antigenicdeterminant is Macrophage colony-stimulating factor (M-CSF or CSF-1).M-CSF or CSF-1 is a regulator of proliferation, differentiation andsurvival of macrophages and their bone-marrow progenitors. The receptorfor M-CSF is a cell surface tyrosine kinase receptor, encoded by theprotooncogene cfms. An elevated expression of M-CSF and its receptor hasbeen associated with poor prognosis in several epithelial cancers suchas breast, uterine and ovarian cancer. Tumor progression has beenstudied in a mouse strain resulting from the crossing of a transgenicmouse susceptible to mammary cancer (PyMT) with a mouse containing arecessive null mutation in csf-1 gene. These mice show attenuated latestage invasive carcinoma and pulmonary metastasis compared to the PyMTmouse (Lin et al., J. Exp. Med. 193:727-739 (2001)). The cause seems tobe the absence of macrophage recruitment to neoplastic tissues.Subcutaneous growth of Lewis lung cancer is also impaired in csf.1 nullmice. It is postulated that the mechanism of macrophage enhancement oftumor growth would be through angiogenic factors, growth factors andproteases produced by the macrophages.

Structural data on the soluble form of M-CSF are available (crystalstructure: Pandit et al., Science 258:1358-62 (1992)), and show thatboth the N- and C-termini of the protein are accessible. However, theN-terminus is close to the site of interaction with the receptor. Inaddition, M-CSF is present both in a soluble and cell surface form,where the transmembrane region is at its C-terminus Therefore, in apreferred embodiment of the present invention, the inventive compositioncomprises an amino acid linker containing a cysteine and being,preferably, added at the C-terminus of M-CSF or fragments thereof, orpreferably at the C-terminus of the soluble form of M-CSF. In furtherpreferred embodiments, the amino acid linker containing a free cysteineis fused to the N-terminus of the sequence corresponding to the sequenceof the processed protein or of the soluble form of the protein, orinserted at the N-terminus of the sequence of the mature form of theprotein or of the soluble form of the protein, C-terminally of thesignal peptide. M-CSF is a dimer, where the two monomers are linked viaan interchain disulfide bridge.

An expression system in E. coli has been described for an N-terminal 149amino acid fragment (functional) of M-CSF (Koths et al., Mol. Reprod.Dev. 46:31-37 (1997)). This fragment of M-CSF, preferably modified asoutlined above, represents a preferred antigenic determinant inaccordance with the invention.

The human sequence is shown in SEQ ID No: 247 (Accession: NP_(—)000748).Further preferred antigenic determinants of the present inventioncomprise the N-terminal fragment consisting of residue 33-181 or 33-185of SEQ ID No: 247, corresponding to the soluble form of the receptor.

The mouse sequence (Accession. NP_(—)031804) is shown in sequence ID No:248. The mature sequence starts at amino acid 33. Thus, a preferredantigenic determinant in accordance with the present invention comprisesamino-acid 33-181 or 33-185.

In another specific embodiment, the antigenic determinant is Resistin(Res). Passive immunization studies were performed with a rabbitpolyclonal antibodies generated against a fusion protein of mouseResistin (mRes) fused to GST, expressed in bacteria. This passiveimmunization lead to improved glucose uptake in an animal obesity/TypeII diabetes model (Steppan et al., Nature 409: 307-12 (2001)).

Resistin (Res) is a 114 aa peptide hormone of approximately 12 KD. Itcontains 11 cysteine of which the most N-terminal one was shown to beresponsible for the dimerisation of the protein and the other 10 arebelieved to be involved in intramolecular disulfide bonds (Banerjee andLazar, J. Biol. Chem. 276: 25970-3 (2001)). Mutation of the firstcysteine to alanine abolishes the dimerisation of mRes.

It was shown, that mRes with a FLAG tag at its C-terminus still remainsactive in an animal model (Steppan et al., Nature 409: 307-12 (2001)),similarly a C-terminally HA tagged (Haemagglutinin tag) version ofresistin was shown to be active in a tissue culture assay (Kim et al.,J. Biol. Chem. 276: 11252-6 (2001)), suggesting that the C-terminus isnot very sensitive to introduced modifications. Thus, in a preferredembodiment, the inventive composition comprises an amino-acid linkercontaining a cysteine residue as second attachment site and being fusedat the C-terminus of the resistin sequence. In further preferredembodiments, the amino acid linker containing a free cysteine is fusedto the N-terminus of the sequence corresponding to the sequence of theprocessed protein, or inserted at the N-terminus of the sequence of themature form of the protein, C-terminally of the signal peptide.

For a preferred embodiment of the present invention, MRes or huRes mayalso be expressed as Fc fusion molecules with a protease cleavage siteinserted between Resistin and the Fc part of the construct, preferablyC-terminally of one or more cysteine residues of the hinge region of theFc part of the fusion protein in a eukaryotic expression system, or morepreferably according to the descriptions and disclosures of EXAMPLE 2.Cleavage of the fusion protein releases Resistin additionally comprisingeither an amino acid linker containing a cysteine residue as describedin EXAMPLE 2, or part or all of the hinge region of the Fc part of thefusion protein which comprises a cysteine residue at its C-terminus,which is suitable for coupling to VLPs or Pili. The human Resistinsequence is shown in SEQ ID No: 249 (Accession AF323081). The mousesequence is shown in SEQ ID No: 250 (Accession AF323080). A favoredembodiment of the invention is human resistin protein fused at itsC-terminus to an amino acid linker containing a cysteine residue. Humanresistin construct can be generated according to the teachings disclosedin EXAMPLE 2, and by comparing murine and human Resistin sequences in aprotein sequence alignment to identify the part of the sequence of humanResistin to be cloned in the vectors described in EXAMPLE 1 and EXAMPLE2 according to the teachings of EXAMPLE 2, or in other suitableexpression vectors. Example of human resistin constructs suitable forgenerating compositions of the inventions are human resistin-C-Xa: (SEQID NO:325), human resistin-C-EK: (SEQ ID NO:326) and human resistin-C:(SEQ ID NO:327).

Human Resistin constructs so generated are a preferred embodiment of theinvention. Vaccination against Resistin using the aforementionedcompositions of the invention may thus provide a way of treating Type IIDiabetes and obesity.

In another embodiment the antigenic determinant is Lymphotoxin-β.Immunization against lymphotoxin-13 may be useful in treating Prionmediated disease. Scrapie (a prion-mediated disease) agent replicationis believed to take mainly place in lymphoid tissues and was shown todepend on prion-protein expressing follicular dendritic cells (FDCs)(Brown et al., Nature Med. 11: 1308-1312 (1999)). It was subsequentlyshown that mice lacking functional follicular dendritic cells show animpaired prion replication in spleens and a (small) retardation ofneuroinvasion (Montrasio et al., Science 288: 1257-1259 (2000)). Thiswas achieved by injecting the mice with a soluble lymphotoxin-βreceptor-Fc-fusion protein (LTβR-Fc). This soluble receptor constructinhibits the development of FDCs by interfering with the crucialinteraction of lymphotoxin-β on T, B or NK cells with the lymphotoxin-βreceptor on the FDC precursor cells. Thus, vaccination againstlymphotoxin-β (also called TNFγ) may provide a vaccine for treatment orprevention of Creutzfeld-Jakob (variant form) or other prion-mediateddiseases and thus prevent prion replication and neuroinvasion.

Immunization against Lymphotoxin-β may also provide a way of treatingdiabetes. Transgene expression of soluble LTβR-Fc fusion protein innonobese diabetic NOD mice blocked diabetes development but notinsulitis (Ettinger et al., J. Exp. Med. 193: 1333-40 K (2001)). Wu etal. (J. Exp. Med. 193: 1327-32 (2001)) also used NOD mice to study theinvolvement of lymphotoxin-β, but instead of transgenic animals they didinject the LTβR-Fc fusion protein. They saw a strong inhibition ofdiabetes development and inhibition of insulitis. Most interestingly,they could even reverse preexisting insulitis by the fusion proteintreatment. In the pancreas the formation of lymphoid follicularstructures could thus be reversed. Vaccination against lymphotoxin-β maythus provide a way of treatment against type-I diabetes.

The sequence of the extracellular domain of human lymphotoxin-β is shownin SEQ ID No: 250 (TNFC_human) and the sequence of the extracellulardomain of murine lymphotoxin-β is shown in SEQ ID No: 251 (TNFC_mouse).

In a further preferred embodiment, the inventive composition comprisesan amino acid linker containing a free cysteine and being added to theN-terminus of the sequence corresponding to the processed form oflymphotoxin-β, or inserted between the N-terminus of the sequencecorresponding to the mature form of the protein, and the signal peptide,C-terminally to the signal peptide. In further preferred embodiments ofthe invention, the extracellular part of lymphotoxin-β is expressed as afusion protein either with Glutathion-S-transferase, fused N-terminallyto lymphotoxin-β, or with a 6 histidine-tag followed by a myc-tag, fusedagain N-terminally to the extracellular part of lymphotoxin-β. An aminoacid spacer containing a protease cleavage site as well as a linkersequence containing a free cysteine as attachment site, C-terminally tothe protease cleavage site, are fused to the N-terminus of the sequenceof the extracellular part of lymphotoxin-β. Preferably, theextracellular part of lymphotoxin-β consists of fragments correspondingto amino acids 49-306 or 126-306 of lymphotoxin-β. These specificcompositions of the invention may be cloned and expressed in the pCEP-Pueukaryotic vector. In further preferred embodiments, the inventivecompositions comprise an amino acid linker containing a free cysteineresidue suitable as second attachment site, and being fused to theC-terminus of lymphotoxin-β or lymphotoxin-β fragments. In aparticularly favored embodiment, the amino acid sequence LACGG (SEQ IDNO:415), comprising the amino acid linker ACGG (SEQ ID NO:416) whichitself contains a cysteine residue for coupling to VLPS and Pili isfused to the N-terminus of the extracellular part of lymphotoxin-β: orof a fragment of the extracellular part of lymphotoxin-β, yielding theproteins human C-LTβ₄₉₋₃₀₆ (SEQ ID NO:346) and human C-LTβ₁₂₆₋₃₀₆ (SEQID NO:347) after cleavage with enterokinase of the corresponding fusionproteins expressed either in vector pCEP-SP-GST-EK or vectorpCP-SP-his-myc-EK as described in EXAMPLE 3.

In a preferred embodiment, the antigen or antigenic determinant is theprion protein, fragments thereof and in particular peptides of the prionprotein. In one embodiment the prion protein is the human prion protein.Guidance on how to modify human prion protein for association with thecore particle is given throughout the application and in particular inEXAMPLE 7. Mouse prion protein constructs are disclosed, and human prionprotein constructs can also be generated and have, for example, thesequence of SEQ ID NO: 348. Further constructs comprise the whole humanprion protein sequence, and other fragments of the human prion protein,which are further composition of the invention. Immunization againstprion protein may provide a way of treatment or prevention ofCreutzfeldt-Jakob (variant form) or other prion-mediated diseases.Immunization using the compositions of the invention comprising theprion protein may provide a way of treatment against prion mediateddiseases in other animals, and the corresponding sequences of bovine andsheep prion protein constructs are given in SEQ ID NO:349 and SEQ IDNO:350, respectively. The peptides of the human prion proteincorresponding to the murine peptides described in EXAMPLE 8, and ofamino acid sequence CSAMSRPIIHFGSDYEDRYYRENMHR (“human cprplong”) (SEQID NO:356) and CGSDYEDRYYRENMHR (“human cprpshort”) (SEQ ID NO:357) leadto preferred embodiments of the invention. These peptides comprise anN-terminal cysteine residue added for coupling to VLPs and Pili.Corresponding bovine and sheep peptides are CSAMSRPLIHFGNDYEDRYYRENMHR(“bovine cprplong”) (SEQ ID NO:401) and CGNDYEDRYYRENMHR (“bovinecprpshort”) (SEQ ID NO:402) CSAMSRPLIHFGNDYEDRYYRENMYR (“sheepcprplong”) (SEQ ID NO:403) and CGNDYEDRYYRENMYR (“sheep cprpshort”) (SEQID NO:404), all leading to embodiments of the invention.

In a further preferred embodiment of the invention, the antigenicdeterminant is tumor necrosis factor α (TNF-α), fragments thereof orpeptides of TNF-α. In particular, peptides or fragments of TNF-α can beused to induce a self-specific immune response directed towards thewhole protein by immunizing a human or an animal with vaccines andcompositions, respectively, comprising such peptides or fragments inaccordance with the invention. Preferably, VLPs, bacteriophages orbacterial pili are used as core particle, to which TNF-α, peptides orfragments thereof are attached according to the invention.

The following murine peptides are the murine homologs to human peptidesthat have been shown to be bound by antibodies neutralizing the activityof TNF-α_ (Yone et al. J. Biol. Chem. 270: 19509-19515) and were, in afurther preferred embodiment of the invention, modified with cysteineresidues for coupling to VLPs, bacteriophages or bacterial pili.

MuTNFa peptide: the sequence CGG was added at the N-terminus of theepitope consisting of amino acid residues 22-32 of mature murine TNF-α:CGGVEEQLEWLSQR (SEQ ID NO:388).

3′TNF II peptide: the sequence GGC was fused at the C-terminus of theepitope consisting of amino acid residues 4-22 of mature murine TNF-αand glutamine 21 was mutated to glycine. The sequence of the resultingpeptide is: SSQNSSDKPVAHVVANHGVGGC (SEQ ID NO:359).

5′TNF II peptide: a cysteine residue was fused to the N-terminus of theepitope consisting of amino acid residues 4-22 of mature murine TNF-αand glutamine 21 was mutated to glycine. The sequence of the resultingpeptide is: CSSQNSSDKPVAHVVANHGV (SEQ ID NO:360).

The corresponding human sequence of the 4-22 epitope isSSRTPSDKPVAHVVANPQAEGQ (SEQ ID NO:361). Like for the murine sequence acysteine is, preferably, fused at the N-terminus of the epitope, or thesequence GGC is fused at the C-terminus of the epitope for covalentcoupling to VLPs, bacteriophages or bacterial pili according to theinvention. It is, however, within the scope of the present inventionthat other cysteine containing sequences are fused at the N- orC-termini of the epitopes. In general, one or two glycine residues arepreferably inserted between the added cysteine residue and the sequenceof the epitope. Other amino acids may, however, also be inserted insteadof glycine residues, and these amino acid residues will preferably besmall amino acids such as serine.

The human sequence corresponding to amino acid residues 22-32 isQLQWLNRRANA (SEQ ID NO:362). Preferably, the sequence CGG is fused atthe N-terminus of the epitope for covalent coupling to VLPs or bacterialpili according to the invention. Other TNF-α_epitopes suitable for usingin the present invention have been described and are disclosed forexample by Yone et al. (J. Biol. Chem. 270: 19509-19515).

The invention further includes compositions which contain mimotopes ofthe antigens or antigenic determinants described herein.

The specific composition of the invention comprises an antibody orpreferably an antibody fragment presented on a virus-like particle orpilus for induction of an immune response against said antibody.Antibodies or antibody fragments which are produced by lymphoma cells,may be selected for attachment to the virus-like particle andimmunization, in order to induce a protective immune response againstthe lymphoma.

In other further embodiments, an antibody or antibody fragment mimickingan antigen is attached to the particle. The mimicking antibody orantibody fragment may be generated by immunization and subsequentisolation of the mimicking antibody or antibody fragment by any knownmethod known to the art such as e.g. hybridoma technology (Gherardi, E.et al., J. Immunol. Methods 126: 61-68 (1990)), phage display (Harrisonet al., Methods Enzymol. 267: 83-109 (1996)), ribosome display (Hanes,J. et al., Nat. Biotechnol. 18: 1287-1292 (2000), yeast two-hybrid(Visintin, M. et al., Proc. Natl. Acad. Sci. USA 96: 11723-11728(1999)), yeast surface display (Boder, E T. & Wittrup, K D. Methods.Enzym. 328: 430-444 (2000)), bacterial surface display (Daugherty, P S.et al., Protein Eng. 12: 613-621 (1999)). The mimicking antibody mayalso be isolated from an antibody library or a naïve antibody libraryusing methods known to the art such as the methods mentioned above, forexample.

In a further embodiment, an antibody recognizing the combining site ofanother antibody, i.e. an anti-idiotypic antibody, further called theimmunizing antibody, may be used. The antibody recognized by theanti-idiotypic antibody will be further referred to as the neutralizingantibody. Thus, by immunizing against the anti-idiotypic antibody,molecules with the specificity of the neutralizing antibody aregenerated in situ; we will further refer to these generated antibodiesas the induced antibodies. In another preferred embodiment, theimmunizing antibody is selected to interact with a ligand molecule ofthe target molecule against which immunization is seeked. The ligandmolecule may be any molecule interacting with the target molecule, butwill preferentially interact with the site of the target moleculeagainst which antibodies should be generated for inhibition of itsfunction. The ligand molecule may be a natural ligand of the targetmolecule, or may be any engineered, designed or isolated ligand havingsuitable binding properties.

The immunizing antibodies may be of human origin, such as isolated froma naïve or immune human antibody library, or may have been isolated froma library generated from another animal source, for example of murineorigin.

Coupling of the antibody or antibody fragment to the VLP or pilus isachieved either by limited reduction of exposed disulfide bridges (forexample of the interchain disulfide bridge between CH1 and Cκ or Cλ, ina Fab fragment) or by fusion of a linker containing a free cysteineresidue at the C-terminus of the antibody or antibody fragment. In afurther embodiment, a linker containing a free cysteine residue is fusedto the N-terminus of the antibody or antibody fragment for attachment toa VLP or pilus protein.

A number of vaccine compositions which employ mimotopes are known in theart, as are methods for generating and identifying mimotopes ofparticular epitopes. For example, Arnon et al., Immunology 101:555-562(2000), the entire disclosure of which is incorporated herein byreference, describe mimotope peptide-based vaccines against Schistosomamansoni. The mimotopes uses in these vaccines were obtained by screeninga solid-phase 8mer random peptide library to identify mimotopes of anepitope recognized by a protective monoclonal antibody againstSchistosoma mansoni. Similarly, Olszewska et al., Virology 272:98-105(2000), the entire disclosure of which is incorporated herein byreference, describe the identification of synthetic peptides which mimican epitope of the measles virus fusion protein and the use of thesepeptides for the immunization of mice. In addition, Zuercher et al.,Eur. J. Immunol. 30:128-135 (2000), the entire disclosure of which isincorporated herein by reference, describe compositions and methods fororal anti-IgE immunization using epitope-displaying phage. Inparticular, epitope-displaying M13 bacteriophages are employed ascarriers for an oral anti-IgE vaccine. The vaccine compositions testedcontain mimotopes and epitopes of the monoclonal anti-IgE antibodyBSW17.

The invention thus includes vaccine compositions which contain mimotopesthat elicit immunological responses against particular antigens, as wellas individual mimotope/core particle conjugates and individualmimotope/non-naturally occurring molecular scaffold conjugates whichmake up these vaccine compositions, and the use of these vaccinecompositions to elicit immunological responses against specific antigensor antigenic determinants. Mimotopes may also be polypeptides, such asanti-idiotypic antibodies. Therefore, in a further preferred embodimentof the invention, the antigen or antigenic determinant is ananti-idiotypic antibody or anti-idiotypic antibody fragment.

The invention further includes compositions which contain mimotopes ofthe antigens or antigenic determinants described herein.

Mimotopes of particular antigens may be generated and identified by anynumber of means including the screening of random peptide phage displaylibraries (see, e.g., PCT Publication No. WO 97/31948, the entiredisclosure of which is incorporated herein by reference). Screening ofsuch libraries will often be performed to identify peptides which bindto one or more antibodies having specificity for a particular antigen.

Mimotopes suitable for use in vaccine compositions of the invention maybe linear or circular peptides. Mimotopes which are linear or circularpeptides may be linked to non-natural molecular scaffolds or coreparticles by a bond which is not a peptide bond.

As suggested above, a number of human IgE mimotopes and epitopes havebeen identified which elicit immunological responses against human IgEmolecules. (See, e.g., PCT Publication No. WO 97/31948.) Thus, incertain embodiments, vaccine compositions of the invention includecompositions which elicit an immunological response against immunoglobinmolecules (e.g., IgE molecules).

Peptides which can be used to elicit such immunological responsesinclude proteins, protein subunits, domains of IgE molecules, andmimotopes which are capable of eliciting production of antibodies havingspecificity for IgE molecules. Generally, portions of IgE molecules usedto prepare vaccine compositions will be derived from IgE molecules ofthe species from which the composition is to be administered. Forexample, a vaccine composition intended for administration to humanswill often contain one or more portions of the human IgE molecule,and/or one or more mimotopes which are capable of elicitingimmunological responses against human IgE molecules.

In specific embodiments, vaccine compositions of the invention intendedfor administration to humans will contain at least one portion of theconstant region of the IgE heavy chain set out in SEQ ID NO:176;Accession No. AAB59424 (SEQ ID NO: 176). In more specific embodiments,IgE peptides used to prepare vaccine compositions of the inventioncomprise, or alternatively consist of, peptides having the followingamino acid sequences: CGGVNLTWSRASG (SEQ ID NO:178).

In additional specific embodiments, vaccine compositions of theinvention will contain at least one mimotope which is capable ofeliciting an immune response that results in the production ofantibodies having specificity for a particular antigen.

Examples of mimotopes of IgE suitable for use in the preparation ofvaccine compositions of the invention include peptides having thefollowing amino acid sequences:

SEQ ID SEQ ID Mimotope NO Mimotope NO INHRGYWV 179 VKLPWRFYQV 187RNHRGYWV 180 VWTACGYGRM 188 RSRSGGYWLW 181 GTVSTLS 189 VNLTWSRASG 182LLDSRYW 190 C_(ε)H₃ epitope QPAHSLG 191 VNLPWSRASG 183 LWGMQGR 192VNLTWSFGLE 184 LTLSHPHWVLNHFVS 193 VNLPWSFGLE 185 SMGPDQTLR 194C_(ε)H₃ mimotope VNLTWS 195 VNRPWSFGLE 186 GEFCINHRGYWVCGDPA 216

C. Preparation of the AlphaVaccine Particles

The invention provides novel compositions and methods for theconstruction of ordered and repetitive antigen arrays. As one of skillin the art would know, the conditions for the assembly of the orderedand repetitive antigen array depend to a large extent on the specificchoice of the first attachment site of the non-natural molecularscaffold and the specific choice of the second attachment site of theantigen or antigenic determinant. Thus, practitioner choice in thedesign of the composition (i.e., selection of the first and secondattachment sites, antigen and non-natural molecular scaffold) willdetermine the specific conditions for the assembly of the AlphaVaccineparticle (the ordered and repetitive antigen array and non-naturalmolecular scaffold combined). Information relating to assembly of theAlphaVaccine particle is well within the working knowledge of thepractitioner, and numerous references exist to aid the practitioner(e.g., Sambrook, J. et al., eds., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989); Ausubel, F. et al., eds., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997); Celis, J., ed.,CELL BIOLOGY, Academic Press, 2^(nd) edition, (1998); Harlow, E. andLane, D., “Antibodies: A Laboratory Manual,” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1988), all of which areincorporated herein by reference.

In a specific embodiment of the invention, the JUN and FOS leucinezipper protein domains are utilized for the first and second attachmentsites of the invention, respectively. In the preparation of AlphaVaccineparticles, antigen must be produced and purified under conditions topromote assembly of the ordered and repetitive antigen array onto thenon-natural molecular scaffold. In the particular JUN/FOS leucine zipperprotein domain embodiment, the FOS-antigen or FOS-antigenic determinantshould be treated with a reducing agent (e.g., Dithiothreitol (DTT)) toreduce or eliminate the incidence of disulfide bond formation (Example15).

For the preparation of the non-natural molecular scaffold (i.e.,recombinant Sinbis virus) of the JUN/FOS leucine zipper protein domainembodiment, recombinant E2-JUN viral particles should be concentrated,neutralized and treated with reducing agent (see Example 16).

Assembly of the ordered and repetitive antigen array in the JUN/FOSembodiment is done in the presence of a redox shuffle. E2-JUN viralparticles are combined with a 240 fold molar excess of FOS-antigen orFOS-antigenic determinant for 10 hours at 4[[^(α)]]° C. Subsequently,the AlphaVaccine particle is concentrated and purified by chromatography(Example 16).

1 In another embodiment of the invention, the coupling of thenon-natural molecular scaffold to the antigen or antigenic determinantmay be accomplished by chemical cross-linking. In a specific embodiment,the chemical agent is a heterobifunctional cross-linking agent such as

-maleimidocaproic acid N-hydroxysuccinimide ester (Tanimori et al., J.Pharm. Dyn. 4:812 (1981); Fujiwara et al., J. Immunol. Meth. 45:195(1981)), which contains (1) a succinimide group reactive with aminogroups and (2) a maleimide group reactive with SH groups. A heterologousprotein or polypeptide of the first attachment site may be engineered tocontain one or more lysine residues that will serve as a reactive moietyfor the succinimide portion of the heterobifunctional cross-linkingagent. Once chemically coupled to the lysine residues of theheterologous protein, the maleimide group of the heterobifunctionalcross-linking agent will be available to react with the SH group of acysteine residue on the antigen or antigenic determinant. Antigen orantigenic determinant preparation in this instance may require theengineering of a cysteine residue into the protein or polypeptide chosenas the second attachment site so that it may be reacted to the freemaleimide function on the cross-linking agent bound to the non-naturalmolecular scaffold first attachment sites. Thus, in such an instance,the heterobifunctional cross-linking agent binds to a first attachmentsite of the non-natural molecular scaffold and connects the scaffold toa second binding site of the antigen or antigenic determinant.

3. Compositions, Vaccines, and the Administration Thereof, and Methodsof Treatment

The invention provides vaccine compositions which may be used forpreventing and/or attenuating diseases or conditions. The inventionfurther provides vaccination methods for preventing and/or attenuatingdiseases or conditions in individuals.

In one embodiment, the invention provides vaccines for the prevention ofinfectious diseases in a wide range of species, particularly mammalianspecies such as human, monkey, cow, dog, cat, horse, pig, etc. Vaccinesmay be designed to treat infections of viral etiology such as HIV,influenza, Herpes, viral hepatitis, Epstein Bar, polio, viralencephalitis, measles, chicken pox, etc.; or infections of bacterialetiology such as pneumonia, tuberculosis, syphilis, etc.; or infectionsof parasitic etiology such as malaria, trypanosomiasis, leishmaniasis,trichomoniasis, amoebiasis, etc.

In another embodiment, the invention provides vaccines for theprevention of cancer in a wide range of species, particularly mammalianspecies such as human, monkey, cow, dog, cat, horse, pig, etc. Vaccinesmay be designed to treat all types of cancer: lymphomas, carcinomas,sarcomas, melanomas, etc.

In another embodiment of the invention, compositions of the inventionmay be used in the design of vaccines for the treatment of allergies.Antibodies of the IgE isotype are important components in allergicreactions. Mast cells bind IgE antibodies on their surface and releasehistamines and other mediators of allergic response upon binding ofspecific antigen to the IgE molecules bound on the mast cell surface.Inhibiting production of IgE antibodies, therefore, is a promisingtarget to protect against allergies. This should be possible byattaining a desired T helper cell response. T helper cell responses canbe divided into type 1 (T_(H)1) and type 2 (T_(H)2) T helper cellresponses (Romagnani, Immunol. Today 18:263-266 (1997)). T_(H)1 cellssecrete interferon-gamma and other cytokines which trigger B cells toproduce IgG1-3 antibodies. In contrast, a critical cytokine produced byT_(H)2 cells is IL-4, which drived B cells to produce IgG4 and IgE. Inmany experimental systems, the development of T_(H)1 and T_(H)2responses is mutually exclusive since T_(H)1 cells suppress theinduction of T_(H)2 cells and vice versa. Thus, antigens that trigger astrong T_(H)1 response simultaneously suppress the development of T_(H)2responses and hence the production of IgE antibodies. Interestingly,virtually all viruses induce a T_(H)1 response in the host and fail totrigger the production of IgE antibodies (Coutelier et al., J. Exp. Med.165:64-69 (1987)). This isotype pattern is not restricted to liveviruses but has also been observed for inactivated or recombinant viralparticles (Lo-Man et al., Eur. J. Immunol. 28:1401-1407 (1998)). Thus,by using the processes of the invention (e.g., AlphaVaccine Technology),viral particles can be decorated with various allergens and used forimmunization. Due to the resulting “viral structure” of the allergen, aT_(H)1 response will be elicited, “protective” IgG1-3 antibodies will beproduced, and the production of IgE antibodies which cause allergicreactions will be prevented. Since the allergen is presented by viralparticles which are recognized by a different set of helper T cells thanthe allergen itself, it is likely that the allergen-specific IgG1-3antibodies will be induced even in allergic individuals harboringpre-existing T_(H)2 cells specific for the allergen. The presence ofhigh concentrations of IgG antibodies may prevent binding of allergensto mast cell bound IgE, thereby inhibiting the release of histamine.Thus, presence of IgG antibodies may protect from IgE mediated allergicreactions. Typical substances causing allergies include: grass, ragweed,birch or mountain cedar pollens, house dust, mites, animal danders,mold, insect venom or drugs (e.g., penicillin). Thus, immunization ofindividuals with allergen-decorated viral particles should be beneficialnot only before but also after the onset of allergies.

In specific embodiments, the invention provides methods for preventingand/or attenuating diseases or conditions which are caused orexacerbated by “self” gene products (e.g., tumor necrosis factors), i.e.“self antigens” as used herein. In related embodiments, the inventionprovides methods for inducing immunological responses in individualswhich lead to the production of antibodies that prevent and/or attenuatediseases or conditions are caused or exacerbated by “self” geneproducts. Examples of such diseases or conditions include graft versushost disease, IgE-mediated allergic reactions, anaphylaxis, adultrespiratory distress syndrome, Crohn's disease, allergic asthma, acutelymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), Graves'disease, inflammatory autoimmune diseases, myasthenia gravis, systemiclupus erythematosus (SLE), immunoproliferative disease lymphadenopathy(IPL), angioimmunoproliferative lymphadenopathy (AIL), immunoblastivelymphadenopathy (IBL), rheumatoid arthritis, diabetes, multiplesclerosis, osteoporosis and Alzheimer's disease.

As would be understood by one of ordinary skill in the art, whencompositions of the invention are administered to an individual, theymay be in a composition which contains salts, buffers, adjuvants, orother substances which are desirable for improving the efficacy of thecomposition. Examples of materials suitable for use in preparingpharmaceutical compositions are provided in numerous sources includingREMINGTON'S PHARMACEUTICAL SCIENCES (Osol, A, ed., Mack Publishing Co.,(1990)).

Compositions of the invention are said to be “pharmacologicallyacceptable” if their administration can be tolerated by a recipientindividual. Further, the compositions of the invention will beadministered in a “therapeutically effective amount” (i.e., an amountthat produces a desired physiological effect).

The compositions of the present invention may be administered by variousmethods known in the art, but will normally be administered byinjection, infusion, inhalation, oral administration, or other suitablephysical methods. The compositions may alternatively be administeredintramuscularly, intravenously, or subcutaneously. Components ofcompositions for administration include sterile aqueous (e.g.,physiological saline) or non-aqueous solutions and suspensions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Carriers or occlusive dressings can be used to increaseskin permeability and enhance antigen absorption.

Prion-mediated diseases are an increasing threat for society.Specifically, prion-induced BSE in cattle represents a disease that haslong been neglected and may affect a great number of animals throughoutEurope. Moreover, a variant form of CJD is attributed to infection ofhumans after consumption of meat of prion-infected cattle. Although thenumber of infected people has been relatively low so far, it seemspossible that the disease may become epidemic. However, long-termprognosis for the development of vCJD may be particular difficult, sinceincubation times between infection and overt disease are very long (anestimated 10 years).

Prions are cellular proteins existing in most mammalian species. Prionproteins exist in two forms, a normally folded form that is usuallypresent in healthy individuals (PrP^(c)) and a misfolded form thatcauses disease (Prp^(Sc)). The current prion hypotheses postulates thatthe misfolded prion form Prp^(Sc) can catalyse the refolding of healthyprion PrP^(c) into disease causing Pip^(Sc) (A. Aguzzi, Haematologica85, 3-10 (2000)). In some rare instances, this transition may also occurspontaneously, causing classical CJD in humans. Some mutations inPrP^(c) are associated with an increase in this spontaneous transition,causing the various forms of familial CJD. However, Prp^(Sc) may also beinfectious and may be transmitted by blood transfusion or via the foodchain. The latter form of prion mediated disease is known as Kuru Kuruand used to occur in human cannibals. However, since species that arefeeding on their own individuals are not abundant, this form of orallytransmitted disease was too rare to be documented for other species.

The massive feeding of cows with beef-products throughout Europe nowchanged the situation and numbers of cows infected with a transmissibleform of BSE-causing Prp^(Sc), dramatically increased in recent years,afflicting hundreds of thousands of cows. This sudden appearance ofmassive numbers of BSE-diseased cows caused great fear in the humanpopulation that a similar disease may be induced in humans. Indeed, in1996, the first case of a variant form of CJD was reported that could beattributed to the consumption of Prp^(Sc) infected beef Until now, thisfear has further increased, since the number of infected humans hasconstantly increased during the following years and no cure is in sight.Moreover, since sheep succumb to a prion-mediated disease called scrapieand since other mammalian species can be infected with Prp^(Sc)

Experimentally, it is possible that BSE-like diseases may occur also inother species. The mechanism of prion transmission has been studied ingreat detail. It is now clear that prions first replicate in thelymphoid organs of infected mice and are subsequently transported to thecentral nervous system. Follicular dendritic cells (FDCs), a rare cellpopulation in lymphoid organs, seems to be essential for bothreplication of prion proteins in the lymphoid organs and transport intothe central nervous system (S. Brandner, M. A. Klein, A. Aguzzi,Transfus Clin Biol 6, 17-23 (1999); F. Montrasio, et al., Science 288,1257-9 (2000)). FDCs are a poorly studied cell type but it is now clearthat they depend upon the production of lymphotoxin and/or TNF by Bcells for their development (F. Mackay, J. L. Browning, Nature 395,26-27 (1998)). Indeed, mice deficient for lymphotoxin do not exhibitFDCs (M. S. Matsumoto, et al., Science 264, 703-707 (1996)). Moreover,they fail to be productively infected with prions and do not succumb todisease. In addition to FDCs, antibodies may also play a role in diseaseprogression (S. Brandner, M. A. Klein, A. Aguzzi, Transfus Clin Biol 6,17-23 (1999)).

Recently, it was shown that blocking the LTb pathway using a Ltbreceptor Fc fusion molecule not only eliminates FDCs in mice but alsoblocks infection with PrP^(Sc) (F. Montrasio, et al., Science 288,1257-9 (2000). Thus, a vaccine that induces antibodies specific for LTbor its receptor may be able to block transmission of PrP^(Sc) from oneindividual to another or from the periphery to the central nervoussystem.

However, it is usually difficult if not impossible to induce antibodyresponses to self-molecules by conventional vaccination. One way toimprove the efficiency of vaccination is to increase the degree ofrepetitiveness of the antigen applied: Unlike isolated proteins, virusesinduce prompt and efficient immune responses in the absence of anyadjuvants both with and without T-cell help (Bachmann & Zinkemagel, Ann.Rev. Immunol: 15:235-270 (1991)). Although viruses often consist of fewproteins, they are able to trigger much stronger immune responses thantheir isolated components. For B-cell responses, it is known that onecrucial factor for the immunogenicity of viruses is the repetitivenessand order of surface epitopes. Many viruses exhibit a quasi-crystallinesurface that displays a regular array of epitopes which efficientlycrosslinks epitope-specific immunoglobulins on B cells (Bachmann &Zinkernagel, Immunol. Today 17:553-558 (1996)). This crosslinking ofsurface immunoglobulins on B cells is a strong activation signal thatdirectly induces cell-cycle progression and the production of IgMantibodies. Further, such triggered B cells are able to activate Thelper cells, which in turn induce a switch from IgM to IgG antibodyproduction in B cells and the generation of long-lived B cell memory—thegoal of any vaccination (Bachmann & Zinkernagel, Ann. Rev. Immunol.15:235-270 (1997)). Viral structure is even linked to the generation ofanti-antibodies in autoimmune disease and as a part of the naturalresponse to pathogens (see Fehr, T., et al., J Exp. Med. 185:1785-1792(1997)). Thus, antibodies presented by a highly organized viral surfaceare able to induce strong anti-antibody responses.

The immune system usually fails to produce antibodies againstself-derived structures. For soluble antigens present at lowconcentrations, this is due to tolerance at the Th cell level. Underthese conditions, coupling the self-antigen to a carrier that candeliver T help may break tolerance. For soluble proteins present at highconcentrations or membrane proteins at low concentration, B and Th cellsmay be tolerant. However, B cell tolerance may be reversible (anergy)and can be broken by administration of the antigen in a highly organizedfashion coupled to a foreign carrier (Bachmann & Zinkernagel, Ann. Rev.Immunol. 15:235-270 (1997). Thus, LTb, LTa or LTb receptor as highlyorganized as a virus, a virus like particle or a bacterial pilus may beable to break B cell tolerance and to induce antibodies specific forthese molecules.

The present invention is related to the fields of molecular biology,virology, immunology and medicine. The invention provides a method thatfacilitates induction of antibodies specific for endogenous lymphotoxin(LT)b, LTa or LTb receptor. The invention also provides a process forproducing an antigen or antigenic determinant that is able to elicitantibodies specific for LTb, LTa or LTb receptor which is useful for theprevention and therapy of prion-mediated diseases such as variantCreutzfeld-Jacob disease (vCJD) or bovine spongioform encephalopathy(BSE) and elimination of lymphoid organ like structures in autoimmunediseased tissues.

The object of the invention is to provide a vaccine that is able toinduce antibodies specific for LTb, LTa or LTb receptor therebyeliminating FDCs from lymphoid organs. This treatment may allowpreventing infection with PrP^(Sc) or spread of PrP^(Sc) from theperiphery to the central nervous system. In addition, this treatmentblocks generation of lymphoid organ like structures in organs targetedby autoimmune disease and may even dissolve such existing structures,ameliorating disease symptoms.

LTb, LTa or LTb receptor or fragments thereof are coupled to a proteincarrier that is foreign to the host. In a preferred embodiment of theinvention, LTb, LTa or LTb receptor or fragments thereof will be coupledto a highly organized structure in order to render these moleculeshighly repetitive and organized. The highly organized structure may be abacterial pilus, a virus like particle (VLP) generated by recombinantproteins of the bacteriophage Qβ, recombinant proteins of Rotavirus,recombinant proteins of Norwalkvirus, recombinant proteins ofAlphavirus, recombinant proteins of Foot and Mouth Disease virus,recombinant proteins of Retrovirus, recombinant proteins of Hepatitis Bvirus, recombinant proteins of Tobacco mosaic virus, recombinantproteins of Flock House Virus, and recombinant proteins of humanPapillomavirus. In order to optimize the three-dimensional arrangementof LTb, LTa or LTb receptor or fragments thereof on the highly organizedstructure, an attachment site, such as a chemically reactive amino-acid,is introduced into the highly organized structure (unless it isnaturally there) and a binding site, such as a chemically reactive aminoacid, will be introduced on the LTb, LTa or LTb receptor or fragments(unless it is naturally there). The presence of an attachment site onthe highly organized structure and a binding site on the LTb, LTa or LTbreceptor or fragments thereof will allow to couple these molecules tothe repetitive structure in an oriented and ordered fashion which isessential for the induction of efficient B cell responses.

In an equally preferred embodiment, the attachment site introduced inthe repetitive structure is biotin that specifically binds streptavidin.Biotin may be introduced by chemical modification. LTb, LTa or LTbreceptor or fragments thereof may be fused or linked to streptavidin andbound to the biotinylated repetitive structure.

Other embodiments of the invention include processes for the productionof the compositions of the invention and methods of medical treatmentusing said compositions. It is to be understood that both the foregoinggeneral description and the following detailed description are exemplaryand explanatory only and are intended to provide further explanation ofthe invention as claimed.

In addition to vaccine technologies, other embodiments of the inventionare drawn to methods of medical treatment for cancer and allergies.

All patents and publications referred to herein are expresslyincorporated by reference in their entirety.

EXAMPLES

Enzymes and reagents used in the experiments that follow included: T4DNA ligase obtained from New England Biolabs; Taq DNA Polymerase,QIAprep Spin Plasmid Kit, QIAGEN Plasmid Midi Kit, QiaExII GelExtraction Kit, QIAquick PCR Purification Kit obtained from QIAGEN;QuickPrep Micro mRNA Purification Kit obtained from Pharmacia;SuperScript One-step RT PCR Kit, fetal calf serum (FCS), bacto-tryptoneand yeast extract obtained from Gibco BRL; Oligonucleotides obtainedfrom Microsynth (Switzerland); restriction endonucleases obtained fromBoehringer Mannheim, New England Biolabs or MBI Fermentas; Pwopolymerase and dNTPs obtained from Boehringer Mannheim. HP-1 medium wasobtained from Cell culture technologies (Glattbrugg, Switzerland). Allstandard chemicals were obtained from Fluka-Sigma-Aldrich, and all cellculture materials were obtained from TPP.

DNA manipulations were carried out using standard techniques. DNA wasprepared according to manufacturer instruction either from a 2 mlbacterial culture using the QIAprep Spin Plasmid Kit or from a 50 mlculture using the QIAGEN Plasmid Midi Kit. For restriction enzymedigestion, DNA was incubated at least 2 hours with the appropriaterestriction enzyme at a concentration of 5-10 units (U) enzyme per mgDNA under manufacturer recommended conditions (buffer and temperature).Digests with more than one enzyme were performed simultaneously ifreaction conditions were appropriate for all enzymes, otherwiseconsecutively. DNA fragments isolated for further manipulations wereseparated by electrophoresis in a 0.7 to 1.5% agarose gel, excised fromthe gel and purified with the QiaExII Gel Extraction Kit according tothe instructions provided by the manufacturer. For ligation of DNAfragments, 100 to 200 pg of purified vector DNA were incubated overnightwith a threefold molar excess of the insert fragment at 16° C. in thepresence of 1 U T4 DNA ligase in the buffer provided by the manufacturer(total volume: 10-20 μl). An aliquot (0.1 to 0.5 μl) of the ligationreaction was used for transformation of E. coli XL1-Blue (Stratagene).Transformation was done by electroporation using a Gene Pulser (BioRAD)and 0.1 cm Gene Pulser Cuvettes (BioRAD) at 200 Ohm, 25 μF, 1.7 kV.After electroporation, the cells were incubated with shaking for 1 h in1 ml S.O.B. medium (Miller, 1972) before plating on selective S.O.B.agar.

Example 1 Modular Eukaryotic Expression System for Coupling of Antigensto VLPs

This system was generated in order to add various amino acid linkersequences containing a cysteine residue to antigens for chemicalcoupling to VLPs.

A. Construction of an EBNA Derived Expression System Encoding aCysteine-Containing Amino Acid Linker and Cleavable Fc-Tag:

pCep-Pu (Wuttke et al. J. Biol. Chem. 276: 36839-48 (2001)) was digestedwith Kpn I and Bam HI and a new multiple cloning site was introducedwith the annealed oligonucleotides PH37 (SEQ ID NO:270) and PH38 (SEQ IDNO:271) leading to pCep-MCS.

A modular system containing a free cysteine flanked by several glycines,a protease cleavage site and the constant region of the human IgG1 wasgenerated as follows. pSec2/Hygro B (Invitrogen Cat. No. V910-20) wasdigested with Bsp120I and Hind III and ligated with the annealedoligonucleotides SU7 (SEQ ID NO:278) and SU8 (SEQ ID NO:279) leading toconstruct pSec-B-MCS. pSec-B-MCS was then digested with Nhe I and HindIII and ligated with the annealed oligonucleotides PH29 (SEQ ID NO:264)and PH30 (SEQ ID NO:265) leading to construct pSec 29/30. The constructpSec-FL-EK-Fc* was generated by a three fragment ligation of thefollowing fragments; first pSec 29/30 digested with Eco RI and Hind III,the annealed oligonucleotides PH31 (SEQ ID NO:266) and PH32 (SEQ ID NO.267) and the Bgl I/EcoRI fragment of a plasmid (pSP-Fc*-C1) containing amodified version of the human IgG1 constant region (for details of thehu IgG1 sequence see the sequence of the final construct pCep-Xa-Fc* seeFIG. 1A-1C (SEQ ID NOs:426, 427 and 428, respectively). The completesequence of pCep-Xa-Fc* is given in SEQ ID NO:283. The resultingconstruct was named pSec-FL-EK-Fc*. From this plasmid the linker regionand the human IgG1 Fc part was excised by Nhe I, Pme I digestion andcloned into pCep-MCS digested with Nhe I and Pme I leading to constructpCep-FL-EK-Fc*. Thus a modular vector, was created where the linkersequence and the protease cleavage site, which are located between theNhe I and Hind III sites, can easily be exchanged with annealedoligonucleotides. For the generation of cleavable fusion protein vectorspCep-FL-EK-Fc* was digested with Nhe I and Hind III and the Factor Xacleavage site N-terminally flanked with amino acids GGGGCG (SEQ IDNO:413) was introduced with the annealed oligonuclotides PH35 (SEQ IDNO:268) and PH36 (SEQ ID NO:269) and the enterokinase site flankedn-terminally with GGGGCG (SEQ ID NO:413) was introduced with theannealed oligonucleotides PH39 (SEQ ID NO:272) and PH40 (SEQ ID NO:273)leading to the constructs pCep-Xa-Fc* (see FIG. 1A, (SEQ ID NO:426) andpCep-EK-Fc* (see FIG. 1B, (SEQ ID NO:427) respectively. The constructpCep-SP-EK-Fc* (see FIG. 1C, (SEQ ID NO:428)) which in addition containsa eukaryotic signal peptide was generated by a three fragment ligationof pCep-EK-Fc* digested Kpn I/Bam HI, the annealed oligos PH41 (SEQ IDNO:274) and PH42 (SEQ ID NO:275) and the annealed oligos PH43 (SEQ IDNO:276) and PH44 (SEQ ID NO:277).

B. Large Scale Production of Fusion Proteins:

For the large scale production of the different fusion proteins 293-EBNAcells (Invitrogen) were transfected with the different pCep expressionplasmids with Lipofectamine 2000 reagent (life technologies) accordingto the manufacturer's recommendation. 24-36 h post transfection thecells were split at a 1 to 3 ratio under puromycin selection (1 μg/ml)in DMEM supplemented with 10% FCS. The resistant cells were thenexpanded in selective medium. For the harvesting of the fusion proteinsthe resistant cell population were passed onto poly-L-lysine coateddishes. Once the cells had reached confluence, they were washed 2 timeswith PBS and serum free medium (DMEM) was added to the plates. Thetissue culture supernatant were harvested every 2 to 4 days and replacedwith fresh DMEM medium during a period of up to one month. The harvestedsupernatants were kept at 4° C.

C. Purification of the Fusion Proteins:

The recombinant Fc-fusion proteins were purified by affinitychromatography using protein A sepharose CL-4B (Amersham PharmaciaBiotech AG). Briefly chromatography columns were packed with 1-3 mlprotein A resin and the tissue culture supernatants containing therecombinant proteins were applied to the column with a peristaltic pumpat a flow rate of 0.5-1.5 ml/min. The column was then washed with 20-50ml PBS. Depending on the fusion protein the protease cleavage wasperformed on the column or the protein was eluted as described below.Recombinant fusion proteins were eluted with a citrate/phosphate buffer(pH 3.8) supplemented with 150 mM NaCl and the fractions containing theprotein were pooled and concentrated with ultrafree centrifugal filters(Millipore).

D. Protease Cleavage of Recombinant Fusion Proteins (Factor Xa,Enterokinase):

Eluted recombinant fusion proteins containing the enterokinase (EK)cleavage site were cleaved using the EKmax system (Invitrogen) accordingto the manufacturer's recommendation. The cleaved Fc part of the fusionprotein was removed by incubation with protein A. The enterokinase wasthen removed with the EK-Away system (Invitrogen) according to themanufacturers recommendation. Similarly fusion proteins containing thefactor Xa (Xa) cleavage site were cleaved using the restriction proteasefactor Xa cleavage and removal kit (Roche) according to themanufacturer's recommendation. The cleaved Fc part was removed byincubation with protein A and the protease was removed with thestreptavidin resin provided with the kit.

The different fusion proteins were concentrated with ultrafreecentrifugal filters (Millipore), quantitated by UV spectrophotometrieand used for subsequent coupling reactions.

FIG. 1A-1C shows partial sequences of the different eukaryoticexpression vectors used. Only the modified sequences are shown.

FIG. 1A: pCep-Xa-Fc*: the sequence is shown from the Bam HI site onwardsand different features are shown above the translated sequence. Thearrow indicates the cleavage site of the factor Xa protease.

FIG. 1B: pCep-EK-Fc*: the sequence is shown from the Bam HI site onwardsand different features are shown above the translated sequence. Thearrow indicates the cleavage site of the enterokinase. The sequencedownstream of the Hind III site is identical to the one shown in FIG.1A.

FIG. 1C: pCep-SP-EK-Fc*: the sequence is shown from the beginning of thesignal peptide on and different features are shown above the translatedsequence. The signal peptide sequence which is cleaved of by the signalpeptidase is shown in bold The arrow indicates the cleavage site of theenterokinase. The sequence downstream of the Hind III site is identicalto the one shown in FIG. 1A.

Example 2 Eukaryotic Expression and Coupling of Mouse Resistin to VLPsand Pili A. Cloning of Mouse Resistin:

Total RNA was isolated from 60 mg mouse adipose tissue using a QiagenRNeasy kit according to the manufacturer's recommendation. The RNA waseluted in 40 μl H₂O. This total RNA was than used for the reversetranscription with an oligo dT primer using the ThermoScript™ RT-PCRSystem (Life Technologies) according to the manufacturer'srecommendation. The sample was incubated at 50° C. for 1 h, heated to85° C. for 5 minutes and treated for 20 minutes at 37° C. with RNAseH.

2 μl of the RT reaction were used for the PCR amplification of mouseresistin. The PCR was performed using Platinium TAQ (Life Technologies)according to the manufacturer's recommendation using primers PH19 (SEQID NO:260) and PH20 (SEQ ID NO:261). Primer PH19 (SEQ ID NO:260)corresponds to positions 58-77 and primer PH20 (SEQ ID NO:261) topositions 454-435 of the mouse Resistin sequence. The PCR mix was firstdenatured at 94° C. for 2 minutes and than 35 cycles were performed asfollows: 30 seconds 94° C., 30 seconds 56° C. and 1 minute 72° C., atthe end the samples were left for 10 minutes at 72° C. The PCR fragmentwas purified and subcloned by TA cloning into the pGEMTeasy vector(Invitrogen) leading to pGEMT-mRes. In order to add appropriaterestriction sites a second PCR was performed on pGEMT-mRes with theprimers PH21 (SEQ ID NO:262) and PH22 (SEQ ID NO. 263) primers using thesame cycling program as described above. The forward primer (PH21 (SEQID NO:262)) contains a Bam HI site and nucleotides 81-102 of the mouseResistin sequence. The reverse primer (PH22 (SEQ ID NO:263)) contains anXba I site and nucleotides 426-406 of the mouse Resistin sequence. Theindicated positions refer to the mouse resistin sequence Gene AccessionNo. AF323080. The PCR product was purified and digested with Bam HI andXba I and subcloned into pcmv-Fc*-C1 digested with Bam HI and Xba Ileading to the construct pcmv-mRes-Fc*.

The Resistin open reading frame was excised from pcmv-Res-Fc* by BamHI/Xba I digestion and cloned into pCep-Xa-Fc* and pCep-EK-Fc* (seeEXAMPLE 1, section B) digested with Bam HI and Nhe I leading to theconstructs pCep-mRes-Xa-Fc* and pCep-mRes-EK-Fc* respectively.

B. Production, Purification and Cleavage of Resistin

pCep-mRes-Xa-Fc* and pCep-mRes-EK-Fc* constructs were then used totransfect 293-EBNA cells for the production of recombinant proteins asdescribed in EXAMPLE 1, section B. The tissue culture supernatants werepurified as described in EXAMPLE 1, section C. The purified proteinswere then cleaved as described in EXAMPLE 1, section D. The resultingrecombinant proteins were termed “resistin-C-Xa” or “Res-C-Xa” and“resistin-C-EK” or “Res-C-EK” according to the vector used (see FIG. 2Aand FIG. 2B).

FIG. 2A and FIG. 2B show sequence of recombinant mouse Resistin proteinsused for expression and further coupling. Res-C-Xa (FIG. 2A) andRes-C-EK (FIG. 2B) are shown as a translated DNA sequences. The resistinsignal sequence which is cleaved upon protein secretion by the signalpeptidase is shown in italic. The amino acid sequences which result formsignal peptidase and specific protease (factor Xa or enterokinase)cleavage are shown bold. The bold sequences correspond to the actualprotein sequence which was used for coupling, i.e. SEQ ID NO:280, SEQ IDNO:281. SEQ ID NO:282 corresponds to an alternative resistin proteinconstruct, which can also be used for coupling to virus-like particlesand pili in accordance with the invention.

C. Coupling of Resistin-C-Xa and Resistin-C-EK to Qβ Capsid Protein

A solution of 0.2 ml of 2 mg/ml Qβ capsid protein in 20 mM Hepes, 150 mMNaCl pH 7.4 was reacted for 30 minutes with 5.6 μl of a solution of 100mM SMPH (Pierce) in DMSO at 25° C. on a rocking shaker. The reactionsolution was subsequently dialyzed twice for 2 hours against 1 L of 20mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. 8 μl of the dialyzed Qβ reactionmixture was then reacted with 32 μl of resistin-C-Xa solution (resultingin a final concentration of resistin of 0.39 mg/ml) and 13 μl of the Qβreaction mixture was reacted with 27 μl resistin-C-EK solution(resulting in a final concentration of resistin of 0.67 mg/ml) for fourhours at 25° C. on a rocking shaker. Coupling products were analysed bySDS-PAGE (see FIG. 2C). An additional band of 24 kDa is present in thecoupling reaction, but not in derivatized Qβ and resistin, respectively.The size of 24 kDa corresponds to the expected size of 24 kDa for thecoupled product (14 kDa for Qβ plus 10 kDa for resistin-C-Xa andresistin-C-EK, respectively).

FIG. 2C shows coupling results of resistin-C-Xa and resistin-C-EK to Qβ.Coupling products were analysed on 16% SDS-PAGE gels under reducingconditions. Lane 1: Molecular weight marker. Lane 2: resistin-C-EKbefore coupling. Lane 3: resistin-C-EK-Qβ after coupling. Lane 4: Qβderivatized. Lane 5: resistin-C-Xa before coupling. Lane 6:resistin-C-Xa-Qβ after coupling. Molecular weights of marker proteinsare given on the left margin. Coupled band is indicated by the arrow.

D. Coupling of Resistin-C-Xa and Resistin-C-EK to Fr Capsid Protein

A solution of 0.2 ml of 2 mg/ml fr capsid protein in 20 mM Hepes, 150 mMNaCl pH 7.4 is reacted for 30 minutes with 5.6 μl of a solution of 100mM SMPH (Pierce) in DMSO at 25° C. on a rocking shaker. The reactionsolution is subsequently dialyzed twice for 2 hours against 1 L of 20 mMHepes, 150 mM NaCl, pH 7.4 at 4° C. 8 μl of the dialyzed fr capsidprotein reaction mixture is then reacted with 32 μl of resistin-C-Xasolution (resulting in a final concentration of resistin of 0.39 mg/ml)and 13 μl of the fr capsid protein reaction mixture is reacted with 27μl resistin-C-EK solution (resulting in a final concentration ofresistin of 0.67 mg/ml) for four hours at 25° C. on a rocking shaker.Coupling products are analysed by SDS-PAGE under reducing conditions.

E. Coupling of Resistin-C-Xa and Resistin-C-EK to HBcAg-Lys-2Cys-Mut

A solution of 0.2 ml of 2 mg/ml HBcAg-Lys-2cys-Mut in 20 mM Hepes, 150mM NaCl pH 7.2 is reacted for 30 minutes with 5.6 μl of a solution of100 mM SMPH (Pierce) in DMSO at 25° C. on a rocking shaker. The reactionsolution is subsequently dialyzed twice for 2 hours against 1 L of 20 mMHepes, 150 mM NaCl, pH 7.2 at 4° C. 8 μl of the dialyzedHBcAg-Lys-2cys-Mut reaction mixture is then reacted with 32 μl ofresistin-C-Xa solution and 13 μl of the HBcAg-Lys-2cys-Mut reactionmixture is reacted with 27 μl resistin-C-EK solution for four hours at25° C. on a rocking shaker. Coupling products are analysed by SDS-PAGE.

F. Coupling of Resistin-C-Xa and Resistin-C-EK to Pili

A solution of 400 μl of 2.5 mg/ml Type-1 pili of E. coli in 20 mM Hepes,pH 7.4, is reacted for 60 minutes with a 50-fold molar excess ofcross-linker SMPH diluted from a stock solution in DMSO (Pierce) at RTon a rocking shaker. The reaction mixture is desalted on a PD-10 column(Amersham-Pharmacia Biotech). The protein-containing fractions eluatingfrom the column are pooled, and 8 μl of the desalted derivatized piliprotein is reacted with 32 μl of resistin-C-Xa solution and 13 μl of thedesalted derivatized pili protein is reacted with 27 μl resistin-C-EKsolution for four hours at 25° C. on a rocking shaker. Coupling productsare analysed by SDS-PAGE.

Example 3 A. Introduction of Cys-Containing Linkers, Expression andPurification of Mouse Lymphotoxin-β

The extracellular part of mouse lymphotoxin-β (LT-β) was recombinantlyexpressed with a CGG amino acid linker at its N-terminus. The linkercontained one cysteine for coupling to VLP. A long (aa 49-306) and ashort version (aa 126-306) of the protein were fused at their N-terminusto either glutathione S-transferase (GST) or a histidin-myc tag forpurification. An enterokinase (EK) cleavage-site was inserted forcleavage of the tag.

Construction of C-LTβ49-306 and C-LTβ126-306.

Mouse LTβ49-306 was amplified by PCR with oligos 5′LTβ and 3′LTβ from amouse spleen cDNA library inserted into pFB-LIB. For the PCR reaction,0.5 μg of each primer and 200 ng of the template DNA was used in the 50μl reaction mixture (1 unit of PFX Platinum polymerase, 0.3 mM dNTPs and2 mM MgSO₄). The temperature cycles were as follows: 94° C. for 2minutes, followed by 25 cycles of 94° C. (15 seconds), 68° C. (30seconds), 68° C. (1 minute) and followed by 68° C. for 10 minutes. ThePCR product was phosphorylated with T4 Kinase and ligated into pEntry1A(Life technologies) which has been cut with EcoRV and has beendephosphorylated. The resulting plasmid was named pEntry1A-LTβ49-306.

A second PCR reaction was performed with oligos 5′LTβlong-NheI and3′LTβstop-NotI resp. 5′LTβshort-NheI and 3′LTβstop-NotI usingpEntry1A-LTβ49-306 as a template. Oligos 5′LTβlong-NheI and5′LTβshort-NheI had an internal NheI site and contained codons for aCys-Gly-Gly linker and 3′LTβstop-NotI had an internal NotI site andcontained a stop codon. For the second PCR reaction, 0.5 μg of eachprimer and 150 ng of the template DNA was used in the 50 μl reactionmixture (1 unit of PFX Platinum polymerase, 0.3 mM dNTPs and 2 mMMgSO₄). The temperature cycles were as follows: 94° C. for 2 minutes,followed by 5 cycles of 94° C. (15 seconds), 50° C. (30 seconds), 68° C.(1 minute), followed by 20 cycles of 94° C. (15 seconds), 64° C. (30seconds), 68° C. (1 minute) and followed by 68° C. for 10 minutes.

The PCR products were digested with NheI and NotI and inserted intoeither pCEP-SP-GST-EK or pCEP-SP-his-myc-EK (Wuttke et al. J. Biol.Chem. 276: 36839-48 (2001)). Resulting plasmids were namedpCEP-SP-GST-EK-C-LTβ49-306, pCEP-SP-GST-EK-C-LTβ126-306,pCEP-SP-his-myc-EK-C-LTβ49-306, pCEP-SP-his-myc-EK-C-LTβ126-306,respectively. GST stands for glutathione-S-transferase, EK forenterokinase, his for a hexahistidine tag and myc for anti c-mycepitope. The C indicates the CGG linker containing the additionalcysteine.

All other steps were performed by standard molecular biology protocols.

Sequence of the Oligonucleotides:

5′LTβ: (SEQ ID NO: 284) 5′-CTT GGT GCC GCA GGA TCA G-3′ 3′LTβ:(SEQ ID NO: 285) 5′-CAG ATG GCT GTC ACC CCA C-3′ 5′LTβlong-NheI:(SEQ ID NO: 286) 5′-GCC CGC TAG CCT GCG GTG GTC AGG ATC AGG GAC GTC G-3′5′LTβshort-NheI: (SEQ ID NO: 287) 5′-GCC CGC TAG CCT GCG GTG GTT CTC CAGCTG CGG ATT C -3′ 3′LTβstop-NotI (SEQ ID NO: 288)5′-CAA TGA CTG CGG CCG CTT ACC CCA CCA TCA CCG -3′

Expression and Production of GST-EK-C-LTβ₄₉₋₃₀₆, GST-EK-C-LTβ₁₂₆₋₃₀₆,His-Myc-EK-C-LTβ₄₉₋₃₀₆ and His-Myc-EK-C-LTβ₁₂₆₋₃₀₆

The plasmids pCEP-SP-GST-EK-C-LTβ49-306, pCEP-SP-GST-EK-C-LTβ126-306,pCEP-SP-his-myc-EK-C-LTβ49-306 and pCEP-SP-his-myc-EK-C-LTβ126-306 weretransfected into 293-EBNA cells (Invitrogen) for protein production asdescribed in EXAMPLE 1. The resulting proteins were namedGST-EK-C-LTβ₄₉₋₃₀₆, GST-EK-C-LTβ₁₂₆₋₃₀₆, his-myc-EK-C-LTβ₄₉₋₃₀₆ andhis-myc-EK-C-LTβ₁₂₆₋₃₀₆.

The protein sequences of the LTβ fusion proteins were translated fromthe cDNA sequences:

SEQ ID NO: 289 GST-EK-C-LTβ₄₉₋₃₀₆: SEQ ID NO: 290 GST-EK-C-LTβ₁₂₆₋₃₀₆:SEQ ID NO: 291 his-myc-EK-C-LTβ₄₉₋₃₀₆: SEQ ID NO: 292his-myc-EK-C-LTβ₁₂₆₋₃₀₆:

The fusion proteins were analysed on 12% SDS-PAGE gels under reducingconditions. Gels were blotted onto nitrocellulose membranes. Membraneswere blocked, incubated with a monoclonal mouse anti-myc antibody orwith an anti-GST antibody. Blots were subsequently incubated with horseradish peroxidase-conjugated goat anti-mouse IgG or horse radishperoxidase-conjugated rabbit anti-goat IgG. The results are shown inFIG. 3. GST-EK-C-LTβ₄₉₋₃₀₆ and GST-EK-C-LTβ126-306 could be detectedwith the anti-GST antibody at a molecular weight of 62 kDa and 48 kDa,respectively. his-myc-EK-C-LTβ₄₉₋₃₀₆ and his-myc-EK-C-LTβ₁₂₆₋₃₀₆ couldbe detected with the anti-myc antibody at 40-56 kDa and 33-39 kDa,respectively.

FIG. 3A and FIG. 3B show the result of the expression of LTβ fusionproteins. LTβ fusion proteins were analysed on 12% SDS-PAGE gels underreducing conditions. Gels were blotted onto nitrocellulose membranes.Membranes were blocked, incubated either with a monoclonal mouseanti-myc antibody (dilution 1:2000) (FIG. 3A) or with an anti-GSTantibody (dilution 1:2000) (FIG. 3B). Blots were subsequently incubatedwith horse radish peroxidase-conjugated goat anti-mouse IgG (dilutions1:4000) (FIG. 3A) or horse radish peroxidase-conjugated rabbit anti-goatIgG (dilutions 1:4000) (FIG. 3B). A: Lane 1 and 2:his-myc-EK-C-LTβ₁₂₆₋₃₀₆. Lane 3 and 4: his-myc-EK-C-LTβ₄₉₋₃₀₆. B: Lane 1and 2: GST-EK-C-LTβ₁₂₆₋₃₀₆. Lane 3 and 4: GST-EK-C-LTβ₄₉₋₃₀₆. Molecularweights of marker proteins are given on the left margin.

B. Purification of GST-EK-C-LTβ₄₉₋₃₀₆, GST-EK-C-LTβ₁₂₆₋₃₀₆,His-Myc-EK-C-LTβ₄₉₋₃₀₆ and His-Myc-EK-C-LTβ₁₂₆₋₃₀₆

GST-EK-C-LTβ₄₉₋₃₀₆ and GST-EK-C-LTβ₁₂₆₋₃₀₆ are purified onglutathione-sepharose column and his-myc-EK-C-LTβ₄₉₋₃₀₆ andhis-myc-EK-C-LTβ₁₂₆₋₃₀₆ are purified on Ni-NTA sepharose column usingstandard purification protocols. The purified proteins are cleaved withenterokinase and analysed on a 16% SDS-PAGE gel under reducingconditions

C. Coupling of C-LTβ₄₉₋₃₀₆ and C-LTβ₁₂₆₋₃₀₆ to Qβ Capsid Protein

A solution of 120 μM Qβ capsid protein in 20 mM Hepes, 150 mM NaCl pH7.2 is reacted for 30 minutes with a 25 fold molar excess of SMPH(Pierce), diluted from a stock solution in DMSO, at 25° C. on a rockingshaker. The reaction solution is subsequently dialyzed twice for 2 hoursagainst 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzed Qβreaction mixture is then reacted with the C-LTβ₄₉₋₃₀₆ and C-LTβ₁₂₆₋₃₀₆solution (end concentrations: 60 μM Qβ, 60 μM C-LTβ₄₉₋₃₀₆ andC-LTβ₁₂₆₋₃₀₆) for four hours at 25° C. on a rocking shaker. Couplingproducts are analysed by SDS-PAGE.

D. Coupling of C-LTβ₄₉₋₃₀₆ and C-LTβ₁₂₆₋₃₀₆ to Fr Capsid Protein

A solution of 120 μM fr capsid in 20 mM Hepes, 150 mM NaCl pH 7.2 isreacted for 30 minutes with a 25 fold molar excess of SMPH (Pierce),diluted from a stock solution in DMSO, at 25° C. on a rocking shaker.The reaction solution is subsequently dialyzed twice for 2 hours against1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzed fr capsidprotein reaction mixture is then reacted with the C-LTβ₄₉₋₃₀₆ andC-LTβ₁₂₆₋₃₀₆ solution (end concentrations: 60 μM fr, 60 μM C-LTβ₄₉₋₃₀₆and C-LTβ₁₂₆₋₃₀₆) for four hours at 25° C. on a rocking shaker. Couplingproducts are analysed by SDS-PAGE under reducing conditions.

E. Coupling of C-LTβ₄₉₋₃₀₆ and C-LTβ₁₂₆₋₃₀₆ to HBcAg-Lys-2Cys-Mut

A solution of 120 μM HBcAg-Lys-2cys-Mut capsid in 20 mM Hepes, 150 mMNaCl pH 7.2 is reacted for 30 minutes with a 25 fold molar excess ofSMPH (Pierce), diluted from a stock solution in DMSO, at 25° C. on arocking shaker. The reaction solution is subsequently dialyzed twice for2 hours against 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. Thedialyzed HBcAg-Lys-2cys-Mut reaction mixture is then reacted with theC-LTβ₄₉₋₃₀₆ and C-LTβ₁₂₆₋₃₀₆ solution (end concentrations: 60 μMHBcAg-Lys-2cys-Mut, 60 μM C-LTβ₄₉₋₃₀₆ and C-LTβ₁₂₆₋₃₀₆) for four hoursat 25° C. on a rocking shaker. Coupling products are analysed bySDS-PAGE.

F. Coupling of C-LTβ₄₉₋₃₀₆ and C-LTβ₁₂₆₋₃₀₆ to Pili

A solution of 125 μM Type-1 pili of E. coli in 20 mM Hepes, pH 7.4, isreacted for 60 minutes with a 50-fold molar excess of cross-linker SMPH,diluted from a stock solution in DMSO (Pierce), at RT on a rockingshaker. The reaction mixture is desalted on a PD-10 column(Amersham-Pharmacia Biotech). The protein-containing fractions eluatingfrom the column are pooled, and the desalted derivatized pili protein isreacted with the C-LTβ₄₉₋₃₀₆ and C-LTβ₁₂₆₋₃₀₆ solution (endconcentrations: 60 μM pili, 60 μM C-LTβ₄₉₋₃₀₆ and C-LTβ₁₂₆₋₃₀₆) for fourhours at 25° C. on a rocking shaker. Coupling products are analysed bySDS-PAGE under reducing conditions.

Example 4 A. Introduction of Cys-Containing Linkers, Expression,Purification and Coupling of Rat Macrophage Migration Inhibitory FactorMIF to Qβ

Rat macrophage migration inhibitory factor (rMIF) was recombinantlyexpressed with three different amino acid linkers C1, C2 and C3 fused atits C-terminus. Each of the linker contained one cysteine for couplingto VLP.

Construction of rMIF-C1, rMIF-C2, and rMIF-C3.

The MCS of pET22b(+) (Novagen, Inc.) was changed to GTTTAACTTTAAGAAGGAGATATACATATGGATCCGGCTAGCGCTCGAGGGTTTAAACGG CGGCCGCATGCACC byreplacing the original sequence from the NdeI site to XhoI site withannealed oligos primerMCS-1F and primerMCS-1R (annealing in 15 mMTrisHCl pH 8 buffer). The resulting plasmid was termed pMod00, which hadNdeI, BamHI, NheI, XhoI, PmeI and NotI restriction sites in its MCS. Theannealed pair of oligos Bamhis6-EK-Nhe-F and Bamhis6-EKNhe-R and theannealed pair of oligo1F-C-glycine-linker and oligo1R-C-glycine-linkerwere together ligated into BamHI-NotI digested pMod00 plasmid to getpModEC1, which had an N terminal hexahistidine tag, an enterokinasecleavage site and a C-terminal amino acid glycine linker containing onecysteine residue. The annealed pair of oligos Bamhis6-EK-Nhe-F andBamhi6-EKNhe R together with the annealed pair ofoligo1F-C-gamma1-linker and oligo1R-C-gamma1-linker were ligated intoBamHI-NotI digested pMod00 plasmid to get pModEC2, which had an Nterminal hexahistidine tag, an enterokinase cleavage site and aC-terminal γ1 linker, derived from the hinge region of humanimmunoglobulin γ1, containing one cysteine residue. The annealed pair ofoligos Bamhis6-EK-Nhe-F and Bamhis6-EK-Nhe-R, the annealed pair ofoligo1FA-C-gamma3-linker and oligo1RA-C-gamma3-linker, and the annealedpair of oligo1FB-C-gamma3-linker and oligo1RB-C-gamma3-linker weretogether ligated into BamHI-NotI digested pMod00 to get pModEC3, whichhad an N terminal hexahistidine tag, an enterokinase cleavage site and aC terminal γ3 linker, containing one cysteine residue, derived from thehinge region of mouse immunoglobulin γ3.

pBS-rMIF, which contains the rat MIF cDNA, was amplified by PCR witholigos rMIF-F and rMIF-Xho-R. rMIF-F had an internal NdeI site andrMIF-Xho-R had an internal XhoI site. The PCR product was digested withNdeI and XhoI and ligated into pModEC1, pModEC2 and pModEC3 digestedwith the same enzymes. Resulting plasmids were named pMod-rMIF-C1,pMod-rMIF-C2 and pMod-rMIF-C3, respectively.

For the PCR reaction, 15 pmol of each oligo and 1 ng of the template DNAwas used in the 50 μl reaction mixture (2 units of PFX polymerase, 0.3mM dNTPs and 2 mM MgSO₄). The temperature cycles were as follows: 94° C.for 2 minutes, followed by 30 cycles of 94° C. (30 seconds), 60° C. (30seconds), 68° C. (30 seconds) and followed by 68° C. for 2 minutes.

All other steps were performed by standard molecular biology protocols.

Sequence of the Oligonucleotides:

primerMCS-1F: (SEQ ID NO: 293) 5′-TAT GGA TCC GGC TAG CGC TCG AGG GTTTAA ACG GCG GCC GCA T-3′ primerMCS-1R: (SEQ ID NO: 294)5′-TCG AAT GCG GCC GCC GTT TAA ACC CTC GAG CGC TAG CCG GAT CCA-3′Bamhis6-EK-Nhe-F: (SEQ ID NO: 295)5′-GAT CCA CAC CAC CAC CAC CAC CAC GGTTCT GGT GAC GAC GAT GAC AAA GCG CTA GCC C-3′ Bamhis6-EK-Nhe-R:(SEQ ID NO: 296) 5′-TCG AGG GCT AGC GCT TTG TCA TCG TCGTCA CCA GAA CCG TGG TGG TGG TGG TGG TGT G-3′ oligo1F-C-glycine-linker:(SEQ ID NO: 297) 5′-TCG AGG GTG GTG GTG GTG GTT GCG GTTAAT AAG TTT AAA CGC-3′ oligo1R-C-glycine-linker: (SEQ ID NO: 298)5′-GGC CGC GTT TAA ACT TAT TAA CCG CAA CCA CCA CCA CCA CCC-3′oligo1F-C-gamma1-linker: (SEQ ID NO: 299)5′-TCG AGG ATA AAA CCC ACA CCT CTC CGCCGT GTG GTT AAT AAG TTT AAA CGC-3′ oligo1R-C-gamma1-linker:(SEQ ID NO: 300) 5′-GGC CGC GTT TAA ACT TAT TAA CCA CACGGC GGA GAG GTG TGG GTT TTA TCC-3′ oligo1FA-C-gamma3-linker:(SEQ ID NO: 301) 5′-TCG AGC CGA AAC CGT CTA CCC CGC CGG GTT CTT CTG-3′oligo1RA-C-gamma3-linker: (SEQ ID NO: 302)5′-CAC CAC CAG AAG AAC CCG GCG GGG TAG ACG GTT TCG GC-3′oligo2FB-C-gamma3-linker: (SEQ ID NO: 303)5′-GTG GTG CTC CGG GTG GTT GCG GTT AAT AAG TTT AAA CGC-3′oligo2RB-C-gamma3-linker: (SEQ ID NO: 304)5′-GGC CGC GTT TAA ACT TAT TAA CCG CAA CCA CCC GGA G-3′ rMIF-F:(SEQ ID NO: 305) 5′-GGA ATT CCA TAT GCC TAT GTT CAT CGT GAA CAC-3′rMIF-Xho-R: (SEQ ID NO: 306) 5′-CCC GCT CGA GAG CGA AGG TGG AAC CGTTC-3′

Expression and Purification of rMIF-Cs

Competent E. coli BL21 (DE3) cells were transformed with plasmidspMod-rMIF-C1, pMod-rMIF-C2 and pMod-rMIF-C3. Single colonies fromampicillin (Amp)-containing agar plates were expanded in liquid culture(SB with 150 mM MOPS, pH 7.0, 200 ug/ml Amp, 0.5% glucose) and incubatedat 30° C. with 220 rpm shaking overnight. 1 l of SB (150 mM MOPS, pH7.0, 200 ug/ml Amp) was then inoculated 1:50 v/v with the overnightculture and grown to OD600=2.5 at 30° C. Expression was induced with 2mM IPTG. Cells were harvested after overnight culture and centrifuged at6000 rpm. Cell pellet was suspended in lysis buffer (10 mM Na₂HPO₄, 30mM NaCl, 10 mM EDTA and 0.25% Tween-20) with 0.8 mg/ml lysozyme,sonicated and treated with benzonase. 2 ml of the lysate was then runthrough a 20 ml Q XL- and a 20 ml SP XL-column. The proteins rMIF-C1,rMIF-C2 and rMIF-C3 were in the flow through.

The protein sequences of the rMIF-Cs were translated from the cDNAsequences.rMIF-C1: SEQ ID NO:307rMIF-C2: SEQ ID NO:308rMIF-C3: SEQ ID NO:309

Coupling of rMIF-C1 to Qβ Capsid Protein

A solution of 1.48 ml of 6 mg/ml Qβ capsid protein in 20 mM Hepes, 150mM NaCl pH 7.2 was reacted for 30 minutes with 14.8 μl of a SMPH(Pierce) (from a 100 mM stock solution dissolved in DMSO) at 25° C. Thereaction solution was subsequently dialyzed twice for 3 hours against 2l of 20 mM Hepes, 150 mM NaCl, pH 7.0 at 4° C. A solution of 1.3 ml of3.6 mg/ml rMIF-C1 protein in 20 mM Hepes, 150 mM NaCl pH 7.2 was reactedfor 1 hour with 9.6 μl of a TCEP (Pierce) (from a 36 mM stock solutiondissolved in H₂O) at 25° C. 130 μl of the derivatized and dialyzed Qβwas then reacted with 129 μl of reduced rMIF-C1 in 241 μl of 20 mMHepes, 150 mM NaCl, pH 7.0 over night at 25° C.

Coupling of rMIF-C2 to Qβ Capsid Protein

A solution of 0.9 ml of 5.5 mg/ml Qβ capsid protein in 20 mM Hepes, 150mM NaCl pH 7.2 was reacted for 30 minutes with 9 μl of a SMPH (Pierce)(from a 100 mM stock solution dissolved in DMSO) at 25° C. The reactionsolution was subsequently dialyzed twice for 2 hours against 2 l of 20mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. A solution of 850 μl of 5.80mg/ml rMIF-C2 protein in 20 mM Hepes, 150 mM NaCl pH 7.2 was reacted for1 hour with 8.5 μl of a TCEP (Pierce) (from a 36 mM stock solutiondissolved in H₂O) at RT. 80 μl of the derivatized and dialyzed Qβ wasthen reacted with 85 μl of reduced rMIF-C2 in 335 μl of 20 mM Hepes, 150mM NaCl, pH 7.2 over night at 25° C.

Coupling of rMIF-C3 to Qβ Capsid Protein

A solution of 1.48 ml of 6 mg/ml Qβ capsid protein in 20 mM Hepes, 150mM NaCl pH 7.2 was reacted for 30 minutes with 14.8 μl of a SMPH(Pierce) (from a 100 mM stock solution dissolved in DMSO) at 25° C. Thereaction solution was subsequently dialyzed twice for 3 hours against 2l of 20 mM Hepes, 150 mM NaCl, pH 7.0 at 4° C. A solution of 720 μl of5.98 mg/ml rMIF-C3 protein in 20 mM Hepes, 150 mM NaCl pH 7.2 wasreacted for 1 hour with 9.5 μl of a TCEP (Pierce) (from a 36 mM stocksolution dissolved in H₂O) at 25° C. 130 μl of the derivatized anddialyzed Qβ was then reacted with 80 μl of reduced rMIF-C3 in 290 μl of20 mM Hepes, 150 mM NaCl, pH 7.0 over night at 25° C.

All three coupled products were analysed on 16% SDS-PAGE gels underreducing conditions. Gels were either stained with Coomassie BrilliantBlue or blotted onto nitrocellulose membranes. Membranes were blocked,incubated with a polyclonal rabbit anti-Qb antiserum (dilution 1:2000)or a purified rabbit anti-MIF antibody (Torrey Pines Biolabs, Inc.)(dilution 1:2000). Blots were subsequently incubated with horse radishperoxidase-conjugated goat anti-rabbit IgG (dilutions 1:2000). Theresults are shown in FIG. 4A and FIG. 4B. Coupled products could bedetected in the Coomassie-stained gels (FIG. 4A) and by both anti-Qβantiserum and the anti-MIF antibody (FIG. 4B) clearly demonstrated thecovalent coupling of all three rMIF variants to Qβ capsid protein.

FIG. 4A shows the coupling of the MIF constructs to Qβ. Couplingproducts were analysed on 16% SDS-PAGE gels under reducing conditions.The gel was stained with Coomassie Brilliant Blue. Molecular weights ofmarker proteins are given on the left margin.

FIG. 4B shows the coupling of MIF-C1 to Qβ. Coupling products wereanalysed on 16% SDS-PAGE gels under reducing conditions. Lane 1: MIF-C1before coupling Lane 2: derivatized Qβ before coupling. Lane 3-5:Qβ-MIF-C1 Lanes 1-3 were stained with Coomassie Brilliant Blue. Lanes 4and 5 represent western blots of the coupling reaction developed with ananti-MIF antiserum and an anti-Qβ antiserum, respectively. Molecularweights of marker proteins are given on the left margin.

B Immunization of Mice with MIF-C1 Coupled to Qβ Capsid Protein

Female Balb/c mice were vaccinated with MIF-C1 coupled to Qβ capsidprotein without the addition of adjuvants. 25 μg of total protein ofeach sample was diluted in PBS to 200 ul and injected subcutaneously(100 ml on two ventral sides) on day 0 and day 14. Mice were bledretroorbitally on day 31 and their serum was analyzed using aMIF-specific ELISA.

C. ELISA

ELISA plates were coated with MIF-C1 at a concentration of 5 μg/ml. Theplates were blocked and then incubated with serially diluted mouse sera.Bound antibodies were detected with enzymatically labeled anti-mouse IgGantibody. As a control, preimmune serum of the same mice was alsotested. The results are shown in FIG. 4C. There was a clear reactivityof the mouse sera raised against MIF-C1 coupled to Qβ capsid protein,while the pre-immune sera did not react with MIF (FIG. 4C and data notshown). From the dilution series with the antisera against MIF-C1coupled to Qβ capsid protein, a half-maximal titer was reached at1:84000.

Shown on FIG. 4C are the ELISA signals obtained with the sera of themice vaccinated with MIF-C1 coupled to Qβ capsid protein. Female Balb/cmice were vaccinated subcutaneously with 25 μg of vaccine in PBS on day0 and day 14. Serum IgG against MIF-C1 were measured on day 31. As acontrol, pre-immune sera from one of the mice were analyzed. Results forindicated serum dilutions are shown as optical density at 450 nm. Allvaccinated mice made high IgG antibody titers. No MIF-specificantibodies were detected in control (pre-immune mouse).

Example 5 Coupling of rMIF-C1 to Fr Capsid Protein andHBcAg-Lys-2Cys-Mut Capsid Protein

Coupling of rMIF-C1 to Fr Capsid Protein

A solution of 100 μl of 3.1 mg/ml fr capsid protein in 20 mM Hepes, 150mM NaCl pH 7.2 was reacted for 30 minutes with 3 μl of a 100 mM stocksolution of SMPH (Pierce) dissolved in DMSO at 25° C. In a parallelreaction, fr capsid protein was first alkylated using iodoacetamid andthen reacted with SMPH using the same reaction conditions describedabove. The reaction solutions were subsequently dialyzed twice for 2hours against 2 l of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. Asolution of 80 μl of 5.7 mg/ml rMIF-C1 protein in 20 mM Hepes, 150 mMNaCl pH 7.2, was reacted for 1 hour with 1 μl of a 36 mM TCEP (Pierce)stock solution dissolved in H₂O, at 25° C. 50 μl of the derivatized anddialyzed fr capsid protein and 50 μl of the derivatized, alkylated anddialyzed fr capsid protein were then reacted each with 17 μl of reducedrMIF-C1 for two hours at 25° C.

Coupling products were analysed on 16% SDS-PAGE gels (FIG. 5). Anadditional band of the expected size of 27 kDa (rMIF-C1: apparent MW 13kDa, fr capsid protein apparent MW 14 kDa) and 29 kDa (rMIF-C1: apparentMW 13 kDa, HBcAg-lys-2cys-Mut: apparent MW 15 kDa) can be detected inthe coupling reaction but not in the fr capsid protein and rMIF-C1solutions, clearly demonstrating coupling.

Coupling of rMIF-C1 to Hepatitis HBcAg-Lys-2Cys-Mut Capsid Protein:

A solution of 100 μl of 1.2 mg/ml HBcAg-lys-2cys-Mut capsid protein in20 mM Hepes, 150 mM NaCl pH 7.2 was reacted for 30 minutes with 1.4 μlof a SMPH (Pierce) (from a 100 mM stock solution dissolved in DMSO) at25° C. The reaction solution was subsequently dialyzed twice for 2 hoursagainst 21 of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. A solution of 80μl of 5.7 mg/ml rMIF-C1 protein in 20 mM Hepes, 150 mM NaCl, pH 7.2 wasreacted for 1 hour with 1 μl of a TCEP (Pierce) (from a 36 mM stocksolution dissolved in H₂O) at 25° C. 60 μl of the derivatized anddialyzed HBcAg-lys-2cys-Mut capsid protein was then reacted with 20 μlof reduced rMIF-C1 for two hours at 25° C.

Coupling products were analysed on 16% SDS-PAGE gels (FIG. 5) underreducing conditions. An additional band of the expected size of about 28kDa (rMIF-C1: apparent MW 13 kDa, HBcAg-lys-2cys-Mut: apparent MW 15kDa) can be detected in the coupling reaction but not in derivatizedHBcAg-lys-2cys-Mut or rMIF-C1, clearly demonstrating coupling.

The samples loaded on the gel of FIG. 5 were the following:

Lane 1: Molecular weight marker. Lane 2: rMIF-C1 before coupling. Lane3: rMIF-C1-fr capsid protein after coupling. Lane 4: derivatized frcapsid protein. Lane 5: rMIF-C1-fr after coupling to alkylated fr capsidprotein. Lane 6: alkylated and derivatized fr capsid protein. Lane 7:rMIF-HBcAg-lys-2cys-Mut after coupling. Lane 8 and 9: derivatizedHBcAg-lys-2cys-Mut. The gel was stained with Coomassie Brilliant Blue.Molecular weights of marker proteins are given on the left margin.

Example 6 A. Introduction of Amino Acid Linkers Containing a CysteineResidue, Expression and Purification of Mouse RANKL

A fragment of the receptor activator of nuclear factor kappa b ligand(RANKL), which has also been termed osteoclast differentiation factor,osteoprotegerin ligand and tumor necrosis factor-relatedactivation-induced cytokine was recombinantly expressed with anN-terminal linker containing one cysteine for coupling to VLP.

Construction of Expression Plasmid

The C-terminal coding region of the RANKL gene was amplified by PCR witholigos RANKL-UP and RANKL-DOWN. RANKL-UP had an internal ApaI site andRANKL-DOWN had an internal XhoI site. The PCR product was digested withApaI and XhoI and ligated into pGEX-6p1 (Amersham Pharmacia). Theresulting plasmid was named pGEX-RANKL. All steps were performed bystandard molecular biology protocols and the sequence was verified. Theplasmid pGEX-RANKL codes for a fusion protein of a glutathioneS-transferase-Prescission cleavage site-cysteine-containing amino acidlinker-RANKL (GST-PS-C-RANKL). The cysteine-containing amino acid linkerhad the sequence GCGGG. The construct also contains a hexa-histidine tagbetween the cysteine containing amino acid linker and the RANKLsequence.

Oligos:

RANKL-UP: (SEQ ID NO: 316)5′CTGCCAGGGGCCCGGGTGCGGCGGTGGCCATCATCACCACCATCACCAG CGCTTCTCAGGAG-3′RANKL-DOWN: (SEQ ID NO: 317) 5′-CCGCTCGAGTTAGTCTATGTCCTGAACTTTGAAAG-3′(SEQ ID NO: 318) Protein of GST-PS-C-RANKL    and (SEQ ID NO: 319)cDNA sequence of GST-PS-C-RANKL     1M  S  P  I  L  G  Y  W  K  I  K  G  L  V  Q  P  T  R  L  L  L  E  Y  L  E   1atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaa  26E  K  Y  E  E  H  L  Y  E  R  D  E  G  D  K  W  R  N  K  K  F  E  L  G  L  76gaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttg  51E  F  P  N  L  P  Y  Y  I  D  G  D  V  K  L  T  Q  S  M  A  I  I  R  Y  I 151gagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatata  76A  D  K  H  N  M  L  G  G  C  P  K  E  R  A  E  I  S  M  L  E  G  A  V  L 226gctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttg 101D  I  R  Y  G  V  S  R  I  A  Y  S  K  D  F  E  T  L  K  V  D  F  L  S  K  301gatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaag 126L  P  E  M  L  K  M  F  E  D  R  L  C  H  K  T  Y  L  N  G  D  H  V  T  H 376ctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccat 151P  D  F  M  L  Y  D  A  L  D  V  V  L  Y  M  D  P  M  C  L  D  A  F  P  K 451cctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaa 176L  V  C  F  K  K  R  I  E  A  I  P  Q  I  D  K  Y  L  K  S  S  K  Y  I  A 526ttagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagca 201W  P  L  Q  G  W  Q  A  T  F  G  G  G  D  H  P  P  K  S  D  L  E  V  L  F 601tggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggatctggaagttctgttc 226Q  G  P  G  C  G  G  G  H  H  H  H  H  H  Q  R  F  S  G  A  P  A  M  M  E 676cagGGGCCCGGGTGCGGCGGTGGCCATCATCACCACCATCACCAGCGCTTCTCAGGAGCTCCAGCTATGATGGAA 251G  S  W  L  D  V  A  Q  R  G  K  P  E  A  Q  P  F  A  H  L  T  I  N  A  A 751GGCTCATGGTTGGATGTGGCCCAGCGAGGCAAGCCTGAGGCCCAGCCATTTGCACACCTCACCATCAATGCTGCC 276S  I  P  S  G  S  H  K  V  T  L  S  S  W  Y  H  D  R  G  W  A  K  I  S  N 826AGCATCCCATCGGGTTCCCATAAAGTCACTCTGTCCTCTTGGTACCACGATCGAGGCTGGGCCAAGATCTCTAAC 301M  T  L  S  N  G  K  L  R  V  N  Q  D  G  F  Y  X  L  Y  A  N  I  C  F  R 901ATGACGTTAAGCAACGGAAAACTAAGGGTTAACCAAGATGGCTTCTATTACCTGTACGCCAACATTTGCTTTCGG 326H  H  E  T  S  G  S  V  P  T  D  Y  L  Q  L  M  V  Y  V  V  K  T  S  I  K 976CATCATGAAACATCGGGAAGCGTACCTACAGACTATCTTCAGCTGATGGTGTATGTCGTTAAAACCAGCATCAAA 351I  P  S  S  H  N  L  M  K  G  G  S  T  K  N  W  S  G  N  S  E  F  H  F  Y1051ATCCCAAGTTCTCATAACCTGATGAAAGGAGGGAGCACGAAAAACTGGTCGGGCAATTCTGAATTCCACTTTTAT 376S  I  N  V  G  G  F  F  K  L  R  A  G  E  E  I  S  I  Q  V  S  N  P  S  L1126TCCATAAATGTTGGGGGATTTTTCAAGCTCCGAGCTGGTGAAGAAATTAGCATTCAGGTGTCCAACCCTTCCCTG 401 L  D  P  D  Q  D  A  T  X  F  G  A  F  K  V  Q  D  I  D  * 1201CTGGATCCGGATCAAGATGCGACGTACTTTGGGGCTTTCAAAGTTCAGGACATAGACTAACTCGAGCGG

Expression and Purification of C-RANKL

Competent E. coli BL21 (DE3) Gold pLys cells were transformed with theplasmid pGEX-RANKL. Single colonies from kanamycin andchloramphenicol-containing agar plates were expanded in liquid culture(LB medium, 30 μg/ml kanamycin, 50 μg/ml chloramphenicol) and incubatedat 30° C. with 220 rpm shaking overnight. 1 l of LB (with 30 ug/mlkanamycin) was then inoculated 1:100 v/v with the overnight culture andgrown to OD600=1 at 24° C. Expression was induced with 0.4 mM IPTG.Cells were harvested after 16 h and centrifuged at 5000 rpm. Cell pelletwas suspended in lysis buffer (50 mM Tris-HCl, pH=8; 25% sucrose; 1 mMEDTA, 1% NaN₃; 10 mM DTT; 5 mM MgCl₂; 1 mg/ml Lysozyme; 0.4 u/ml DNAse)for 30 min. Then 2.5 volumes of buffer A (50 mM Tris-HCl, pH=8.0; 1%Triton X100; 100 mM NaCl; 0,1% NaN₃; 10 mM DTT; 1 mM PMSF) were addedand incubated at 37° C. for 15 min. The cells were sonicated andpelleted at 9000 rpm for 15 min. The supernatant was immediately usedfor GST-affinity chromatography.

A column GST-Trap FF of 5 ml (Amersham Pharmacia) was equilibrated inPBS, pH 7.3 (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄). Thesupernatant was loaded on the 5 ml GST-Trap FF column and subsequentlythe column was rinsed with 5 column volumes of PBS. The proteinGST-PS-C-RANKL was eluted with 50 mM Tris-HCl, pH=8.0 containing GSH 10mM.

The purified GST-PS-C-RANKL protein was digested using the proteasePreScission (Amersham Pharmacia). The digestion was performed at 37° C.for 1 hour using a molar ratio of 500/1 of GST-PS-C-RANKL toPreScission.

Furthermore, the reaction of protease digestion was buffer exchangedusing a HiPrep 26/10 desalting column (Amersham Pharmacia), thefractions containing the proteins were pooled and immediately used foranother step of GST affinity chromatography using the same conditionsreported before. Purification of C-RANKL was analysed on a SDS-PAGE gelunder reducing conditions, shown in FIG. 6. Molecular weights of markerproteins are given on the left margin of the gel in the figure. The gelwas stained with Coomassie Brilliant Blue. The cleaved C-RANKL ispresent in the flow-through (unbound fraction) while the uncleavedGST-PS-C-RANKL, the cleaved GST-PS and the PreScission remain bound tothe column. C-RANKL protein of the expected size of 22 kDa was obtainedin high purity.

The samples loaded on the gel of FIG. 6 were the following:

Lane 1: Low molecular weight marker. Lanes 2 and 3: the supernatant ofthe cell lysates of the BL21/DE3 cells transformed with the empty vectorpGEX6p1 and pGEX-RANKL respectively, after sixteen hours of inductionwith IPTG 0.4 mM. Lane 4: the purified GST-PS-C-RANKL protein afterGST-Trap FF column. Lane 5: the GST-Trap FF column unbound fraction.Lane 6: the purified GST-PS-C-RANKL protein after the cleavage with thePreScission protease. Lane 7: the unbound fraction of the GST-Trap FFcolumn loaded with the GST-RANKL digestion, which contains the purifiedC-RANKL. Lane 8: the bound fraction of the GST-Trap FF column loadedwith the GST-PS-C-RANKL digestion and eluted with GSH.

B. Coupling of C-RANKL to Qβ Capsid Protein

A solution of 120 μM Qβ capsid in 20 mM Hepes, 150 mM NaCl pH 7.2 isreacted for 30 minutes with a 25 fold molar excess of SMPH (Pierce),diluted from a stock solution in DMSO, at 25° C. on a rocking shaker.The reaction solution is subsequently dialyzed twice for 2 hours against1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzed Qβreaction mixture is then reacted with the C-RANKL solution (endconcentrations: 60 μM Qβ, 60 μM C-RANKL) for four hours at 25° C. on arocking shaker. Coupling products are analysed by SDS-PAGE.

C. Coupling of C-RANKL to Fr Capsid Protein

A solution of 120 μM fr capsid in 20 mM Hepes, 150 mM NaCl pH 7.2 isreacted for 30 minutes with a 25 fold molar excess of SMPH (Pierce),diluted from a stock solution in DMSO, at 25° C. on a rocking shaker.The reaction solution is subsequently dialyzed twice for 2 hours against1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzed fr capsidprotein reaction mixture is then reacted with the C-RANKL solution (endconcentrations: 60 μM fr capsid protein, 60 μM C-RANKL) for four hoursat 25° C. on a rocking shaker. Coupling products are analysed bySDS-PAGE.

D. Coupling of C-RANKL to HBcAg-Lys-2Cys-Mut

A solution of 120 μM HBcAg-Lys-2cys-Mut capsid in 20 mM Hepes, 150 mMNaCl pH 7.2 is reacted for 30 minutes with a 25 fold molar excess ofSMPH (Pierce), diluted from a stock solution in DMSO, at 25° C. on arocking shaker. The reaction solution is subsequently dialyzed twice for2 hours against 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. Thedialyzed HBcAg-Lys-2cys-Mut reaction mixture is then reacted with theC-RANKL solution (end concentrations: 60 μM HBcAg-Lys-2cys-Mut, 60 μMC-RANKL) for four hours at 25° C. on a rocking shaker. Coupling productsare analysed by SDS-PAGE.

E. Coupling of C-RANKL to Pili

A solution of 125 μM Type-1 pili of E. coli in 20 mM Hepes, pH 7.4, isreacted for 60 minutes with a 50-fold molar excess of cross-linker SMPH,diluted from a stock solution in DMSO, at RT on a rocking shaker. Thereaction mixture is desalted on a PD-10 column (Amersham-PharmaciaBiotech). The protein-containing fractions eluating from the column arepooled, and the desalted derivatized pili protein is reacted with theC-RANKL solution (end concentrations: 60 μM pili, 60 μM C-RANKL) forfour hours at 25° C. on a rocking shaker. Coupling products are analysedby SDS-PAGE.

Example 7 A. Introduction of Amino Acid Linker Containing a CysteineResidue, Expression and Purification of a Truncated Form of the MousePrion Protein

A truncated form (aa 121-230) of the mouse prion protein (termedmPrP_(t)) was recombinantly expressed with a GGGGCG amino acid linkerfused at its C-terminus for coupling to VLPs and Pili. The protein wasfused to the N-terminus of a human Fc-fragment for purification. Anenterokinase (EK) cleavage-site was introduced behind the EK cleavagesite to cleave the Fc-part of the fusion protein after purification.

Construction of mPrP_(t)EK-Fc*.

Mouse PrP_(t) was amplified by PCR with the primer 5′PrP-BamHI and3′PrP-NheI using the plasmid pBP^(CMV)PrP-Fc as a template.pBP^(CMV)PrP-Fc contained the wild-type sequence of the mouse prionprotein. 5′PrP-BamHI had an internal BamHI site and contained an ATG and3′PrP-NheI had an internal NheI site.

For the PCR reaction, 0.5 μg of each primer and 200 ng of the templateDNA was used in the 50 μl reaction mixture (1 unit of PFX Platinumpolymerase, 0.3 mM dNTPs and 2 mM MgSO₄). The temperature cycles were asfollows: 94° C. for 2 minutes, followed by 5 cycles of 94° C. (15seconds), 50° C. (30 seconds), 68° C. (45 seconds), followed by 20cycles of 94° C. (15 seconds), 64° C. (30 seconds), 68° C. (45 seconds)and followed by 68° C. for 10 minutes.

The PCR product was digested with BamHI and NheI and inserted intopCEP-SP-EK-Fc* containing the GGGGCG linker sequence at the 5′ end ofthe EK cleavage sequence. The resulting plasmid was namedpCEP-SP-mPrP_(t)-EK-Fc*.

All other steps were performed by standard molecular biology protocols.

Oligos:

Primer 5′PrP-BamHI (SEQ ID NO: 321)5′-CGG GAT CCC ACC ATG GTG GGG GGC CTT GG -3′ Primer 3′PrP-NheI(SEQ ID NO: 322) 5′-CTA GCT AGC CTG GAT CTT CTC CCG -3′

Expression and Purification of mPrP_(t)-EK-Fc*

Plasmid pCEP-SP-mPrP_(t)-EK-Fc* was transfected into 293-EBNA cells(Invitrogen) and purified on a Protein A-sepharose column as describedin EXAMPLE 1.

The protein sequence of the mPrP_(t)-EK-Fc* is identified in SEQ IDNO:323.

mPrP_(t) after cleavage has the sequence as identified in SEQ ID NO:324with the GGGGCG linker at its C-terminus

The purified fusion protein mPrP_(t)-EK-Fc* was cleaved withenterokinase and analysed on a 16% SDS-PAGE gel under reducingconditions before and after enterokinase cleavage. The gel was stainedwith Coomassie Brilliant Blue. The result is shown in FIG. 7. Molecularweights of marker proteins are given on the left margin of the gel inthe figure. The mPrP_(t)-EK-Fc* fusion protein could be detected as a 50kDa band. The cleaved mPrP_(t) protein containing the GGGGCG amino acidlinker fused to its C-terminus could be detected as a broad band between18 and 25 kDa. The identity of mPrP_(t) was confirmed by westernblotting (data not shown). Thus, mPrPt with a C-terminal amino acidlinker containing a cysteine residue, could be expressed and purified tobe used for coupling to VLPs and Pili.

The samples loaded on the gel of FIG. 7 were the following.

Lane 1: Molecular weight marker. Lane 2: mPrP_(t)-EK-Fc* beforecleavage. Lane 3: mPrP_(t) after cleavage.

B. Coupling of mPrP_(t) to Qβ Capsid

A solution of 120 μM Qβ capsid in 20 mM Hepes, 150 mM NaCl pH 7.2 isreacted for 30 minutes with a 25 fold molar excess of SMPH (Pierce),diluted from a stock solution in DMSO, at 25° C. on a rocking shaker.The reaction solution is subsequently dialyzed twice for 2 hours against1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzed Qβreaction mixture is then reacted with the mPrP_(t) solution (endconcentrations: 60 μM Qβ, 60 μM mPrP_(t)) for four hours at 25° C. on arocking shaker. Coupling products are analysed by SDS-PAGE.

C. Coupling of mPrP_(t) to Fr Capsid Protein

A solution of 120 μM fr capsid protein in 20 mM Hepes, 150 mM NaCl pH7.2 is reacted for 30 minutes with a 25 fold molar excess of SMPH(Pierce), diluted from a stock solution in DMSO, at 25° C. on a rockingshaker. The reaction solution is subsequently dialyzed twice for 2 hoursagainst 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzed frreaction mixture is then reacted with the mPrP_(t) solution (endconcentrations: 60 μM fr, 60 μM mPrP_(t)) for four hours at 25° C. on arocking shaker. Coupling products are analysed by SDS-PAGE.

D. Coupling of mPrP_(t) to HBcAg-Lys-2Cys-Mut

A solution of 120 μM HBcAg-Lys-2cys-Mut capsid in 20 mM Hepes, 150 mMNaCl pH 7.2 is reacted for 30 minutes with a 25 fold molar excess ofSMPH (Pierce), diluted from a stock solution in DMSO, at 25° C. on arocking shaker. The reaction solution is subsequently dialyzed twice for2 hours against 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. Thedialyzed HBcAg-Lys-2cys-Mut reaction mixture is then reacted with themPrP_(t) solution (end concentrations: 60 μM HBcAg-Lys-2cys-Mut, 60 μMmPrP_(t)) for four hours at 25° C. on a rocking shaker. Couplingproducts are analysed by SDS-PAGE.

E. Coupling of mPrP_(t) to Pili

A solution of 125 μM Type-1 pili of E. coli in 20 mM Hepes, pH 7.4, isreacted for 60 minutes with a 50-fold molar excess of cross-linker SMPH(Pierce), diluted from a stock solution in DMSO, at RT on a rockingshaker. The reaction mixture is desalted on a PD-10 column(Amersham-Pharmacia Biotech). The protein-containing fractions eluatingfrom the column are pooled, and the desalted derivatized pili protein isreacted with the mPrP_(t) solution (end concentrations: 60 μM pili, 60μM mPrP_(t)) for four hours at 25° C. on a rocking shaker. Couplingproducts are analysed by SDS-PAGE.

Example 8 A. Coupling of Prion Peptides to Qβ Capsid Protein PrionPeptide Vaccines

The following prion peptides were chemically synthesized:CSAMSRPMIHFGNDWEDRYYRENMYR (“cprplong”) and CGNDWEDRYYRENMYR(“cprpshort”), which comprise an added N-terminal cysteine residue forcoupling to VLPs and Pili, and used for chemical coupling to Qβ asdescribed in the following.

A solution of 5 ml of 140 μM Qβ capsid protein in 20 mM Hepes. 150 mMNaCl pH 7.4 was reacted for 30 minutes with 108 μl of a 65 mM solutionof SMPH (Pierce) in H₂O at 25° C. on a rocking shaker. The reactionsolution was subsequently dialyzed twice for 2 hours against 5 L of 20mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. 100 μl of the dialyzed reactionmixture was then reacted either with 1.35 μl of a 2 mM stock solution(in DMSO) of the peptide cprpshort (1:2 peptide/Qβ capsid protein ratio)or with 2.7 μl of the same stock solution (1:1 peptide/Qβ ratio). 1 μlof a 10 mM stock solution (in DMSO) of the peptide cprplong was reactedwith 100 μl of the dialyzed reaction mixture. The coupling reactionswere performed over night at 15° C. in a water bath. The reactionmixtures were subsequently dialyzed 24 h against 2×5 L of 20 mM Hepes,150 mM NaCl, pH 7.4 at 4° C.

The coupled products were centrifuged and supernatants and pellets wereanalysed on 16% SDS-PAGE gels under reducing conditions. Gels werestained with Coomassie Brilliant Blue. The results are shown in FIG. 16.Molecular weights of marker proteins are given on the left margin of thegel in the figure. The bands at a molecular weight between 16.5 and 25kDa clearly demonstrated the covalent coupling of the peptides cprpshortand cprplong to Qβ capsid protein.

The samples loaded on the gel of FIG. 16 A were the following:

Lane 1: purified Qβ capsid protein. Lane 2: derivatized Qβ capsidprotein before coupling. Lanes 3-6: Qβ capsid protein-cprpshortcouplings with a 1:2 peptide/Qβ ratio (lanes 3 and 4) and 1:1 peptide/Qβratio (lanes 5 and 6). Soluble fractions (lanes 3 and 5) and insolublefractions (lanes 4 and 6) are shown.

The samples loaded on the gel of FIG. 16 B were the following:

Lane 1: Molecular weight marker. Lane 2: derivatized Qβ capsid proteinbefore coupling. Lane 3 and 4: Qβ capsid protein-cprplong couplingreactions. Soluble fraction (lane 3) and insoluble fraction (lane 4) areshown.

B. Coupling of Prion Peptides to Fr Capsid Protein

A solution of 120 μM fr capsid protein in 20 mM Hepes, 150 mM NaCl pH7.2 is reacted for 30 minutes with a 10 fold molar excess of SMPH(Pierce)), diluted from a stock solution in DMSO, at 25° C. on a rockingshaker. The reaction solution is subsequently dialyzed twice for 2 hoursagainst 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzed frreaction mixture is then reacted with equimolar concentration of peptidecprpshort or a ration of 1:2 cprplong/fr over night at 16° C. on arocking shaker. Coupling products are analysed by SDS-PAGE.

C. Coupling of Prion Peptides to HBcAg-Lys-2Cys-Mut

A solution of 120 μM HBcAg-Lys-2cys-Mut in 20 mM Hepes, 150 mM NaCl pH7.2 is reacted for 30 minutes with a 10 fold molar excess of SMPH(Pierce)), diluted from a stock solution in DMSO, at 25° C. on a rockingshaker. The reaction solution is subsequently dialyzed twice for 2 hoursagainst 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzedHBcAg-Lys-2cys-Mut reaction mixture is then reacted with equimolarconcentration of peptide cprpshort or a ration of 1:2cprplong/HBcAg-Lys-2cys-Mut over night at 16° C. on a rocking shaker.Coupling products are analysed by SDS-PAGE.

D. Coupling of Prion Peptides to Pili

A solution of 125 μM Type-1 pili of E. coli in 20 mM Hepes, pH 7.4, isreacted for 60 minutes with a 50-fold molar excess of cross-linker SMPH(Pierce), diluted from a stock solution in DMSO, at RT on a rockingshaker. The reaction mixture is desalted on a PD-10 column(Amersham-Pharmacia Biotech). The protein-containing fractions eluatingfrom the column are pooled, and the desalted derivatized pili protein isreacted with the prion peptides in equimolar or in a ratio of 1:2peptide pili over night at 16° C. on a rocking shaker. Coupling productsare analysed by SDS-PAGE.

Example 9 Cloning, Expression and Purification of IL-13 to VLPs and Pili

A. Cloning and Expression of Interleukin 13 (IL-13) with an N-TerminalAmino Acid Linker Containing a Cysteine Residue for Coupling to VLPs andPili

a) Cloning of Mouse IL-13 (HEK-293T) for Expression in Mammalian Cellsas Fc Fusion Protein

The DNA for the cloning of IL-13 was isolated by RT-PCR from in vitroactivated splenocytes, which were obtained as following: CD4+ T cellswere isolated from mouse spleen cells and incubated 3 days in IMDM (+5%FCS+10 ng/ml IL4) in 6 well plates which have been previously coatedwith anti-CD3 and anti-CD28 antibodies. The RNA from these cells wasused to amplify IL13 by one-step RT-PCR (Qiagen one-step PCR kit).Primer XhoIL13-R was used for the reverse transcription of the RNA andthe primers NheIL13-F (SEQ ID NO:338) and XhoIL13-R (SEQ ID NO:339) wereused for the PCR amplification of the IL13 cDNA. Amplified IL13 cDNA wasligated in a pMOD vector using the NheI/XhoI restriction sites (givingthe vector pMODB1-IL13). pMODB1-Il13 was digested BamHI/XhoI and thefragment containing IL13 was ligated in the pCEP-SP-XA-Fc*(Δxho) vector,an analogue of pCEP-SP-XA-Fc* where a XhoI site at the end of the Fcsequence has been removed, which had been previously digested withBamHI/XhoI. The plasmid resulting from this ligation (pCEP-SP-IL13-Fc)was sequenced and used to transfect HEK-293T cells. The resulting IL 13construct encoded by this plasmid had the amino acid sequenceADPGCGGGGGLA fused at the N-terminus of the IL-13 mature sequence. Thissequence comprises the amino acid linker sequence GCGGGGG flanked byadditional amino acids introduced during the cloning procedure. IL13-Fccould be purified with Protein-A resin from the supernatant of the cellstransfected with pCEP-SP-IL13-Fc. The result of the expression is shownon FIG. 17 B (see EXAMPLE 10 for description of the samples). MatureIL-13 fused at its N-terminus with the aforementioned amino acidsequence is released upon cleavage of the fusion protein with Factor-Xa,leading to a protein called hereinafter “mouse C-IL-13-F” and having asequence of SEQ ID NO:328. The result of FIG. 17 B clearly demonstratesexpression of the IL-13 construct.

b) Cloning of Mouse IL-13 (HEK-293T) for Expression in Mammalian Cellswith GST (Glutathion-S-Transferase) Fused at its N-Terminus

The cDNA used for cloning IL-13 with an N-terminal GST originated fromthe cDNA of TH2 actiated T-cells as described above (a.). IL-13 wasamplified from this cDNA using the primers Nhelink1IL13-F andIL13StopXhoNot-R. The PCR product was digested with NheI and XhoI andligated in the pCEP-SP-GST-EK vector previously digested with NheI/XhoI.The plasmid which could be isolated from the ligation (pCEP-SP-GST-IL13)was used to transfect HEK-293T cells. The resulting IL 13 constructencoded by this plasmid had the amino acid sequence LACGGGGG fused atthe N-terminus of the IL-13 mature sequence. This sequence comprises theamino acid linker sequence ACGGGGG flanked by an additional amino acidintroduced during the cloning procedure. The culture supernatant of thecells transfected with pCEP-SP-GST-IL13 contained the fusion proteinGST-IL13 which could be purified by Glutathione affinity chromatographyaccording to standard protocols. Mature IL-13 fused at its N-terminuswith aforementioned amino acid sequence is released upon cleavage of thefusion protein with enterokinase, leading to a protein calledhereinafter “mouse C-IL-13-S” and having a sequence of SEQ ID NO:329.

B. Coupling of Mouse C-IL-13-F, Mouse C-IL-13-S to Qβ Capsid Protein

A solution of 120 μM Qβ capsid in 20 mM Hepes, 150 mM NaCl pH 7.2 isreacted for 30 minutes with a 25 fold molar excess of SMPH (Pierce),diluted from a stock solution in DMSO, at 25° C. on a rocking shaker.The reaction solution is subsequently dialyzed twice for 2 hours against1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzed Qβreaction mixture is then reacted with the mouse C-IL-13-F or mouseC-IL-13-S solution (end concentrations: 60 μM Qβ capsid protein, 60 μMmouse C-IL-13-F or mouse C-IL-13-S) for four hours at 25° C. on arocking shaker. Coupling products are analysed by SDS-PAGE.

C. Coupling of Mouse C-IL-13-F, Mouse C-IL-13-S to Fr Capsid Protein

A solution of 120 μM fr capsid protein in 20 mM Hepes, 150 mM NaCl pH7.2 is reacted for 30 minutes with a 25 fold molar excess of SMPH(Pierce), diluted from a stock solution in DMSO, at 25° C. on a rockingshaker. The reaction solution is subsequently dialyzed twice for 2 hoursagainst 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzed frreaction mixture is then reacted with the mouse C-IL-13-F or mouseC-IL-13-S solution (end concentrations: 60 μM fr capsid protein, 60 μMmouse C-IL-13-F or mouse C-IL-13-S) for four hours at 25° C. on arocking shaker. Coupling products are analysed by SDS-PAGE.

D. Coupling of Mouse C-IL-13-F or Mouse C-IL-13-S Solution toHBcAg-Lys-2Cys-Mut

A solution of 120 μM HBcAg-Lys-2cys-Mut capsid in 20 mM Hepes, 150 mMNaCl pH 7.2 is reacted for 30 minutes with a 25 fold molar excess ofSMPH (Pierce), diluted from a stock solution in DMSO, at 25° C. on arocking shaker. The reaction solution is subsequently dialyzed twice for2 hours against 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. Thedialyzed HBcAg-Lys-2cys-Mut reaction mixture is then reacted with themouse C-IL-13-F or mouse C-IL-13-S solution (end concentrations: 60 μMHBcAg-Lys-2cys-Mut, 60 μM mouse C-IL-13-F or mouse C-IL-13-S) for fourhours at 25° C. on a rocking shaker. Coupling products are analysed bySDS-PAGE.

E. Coupling of Mouse C-IL-13-F or Mouse C-IL-13-S Solution to Pili

A solution of 125 μM Type-1 pili of E. coli in 20 mM Hepes, pH 7.4, isreacted for 60 minutes with a 50-fold molar excess of cross-linker SMPH,diluted from a stock solution in DMSO, at RT on a rocking shaker. Thereaction mixture is desalted on a PD-10 column (Amersham-PharmaciaBiotech). The protein-containing fractions eluating from the column arepooled, and the desalted derivatized pili protein is reacted with themouse C-IL-13-F or mouse C-IL-13-S solution (end concentrations: 60 μMpili, 60 μM mouse C-IL-13-F or mouse C-IL-13-S) for four hours at 25° C.on a rocking shaker. Coupling products are analysed by SDS-PAGE.

Example 10 Cloning and Expression of Interleukin 5 (IL-5) with anN-Terminal Amino Acid Linker Containing a Cysteine Residue for Couplingto VLPs and Pili

A. Cloning of IL-5 for Expression as Inclusion Bodies in E. coli

IL-5 was amplified from an ATCC clone (pmIL5-4G; ATCC number: 37562) byPCR using the following two primers: Spelinker3-F1 (SEQ ID NO:340) andIl5StopXho-R (SEQ ID NO:342). The product of this PCR was used astemplate for a second PCR with the primers SpeNlinker3-F2 (SEQ IDNO:341) and Il5StopXho-R. The insert was digested with SpeI and NotI.This insert was ligated into a pET vector derivative (pMODEC3-8 vector),previously digested with NheI and NotI (not dephosphorylated), andtransformed into E. coli TG1 cells. The IL5 construct generated bycloning into pMODEC3-8 vector contains at its N-terminus ahexa-histidine tag, followed by an enterokinase site, an N-terminalgamma 3 amino acid linker containing a cysteine residue, flankedC-terminally by the sequence AS and N-terminally by the sequence ALV,and the mature form of the IL 5 gene. The protein released by cleavagewith enterokinase is called “mouse C-IL-S-E” (SEQ ID NO:332). PlasmidDNA of resulting clone pMODC6-IL5.2 (also called pMODC6-IL5), whosesequence had been confirmed by DNA sequencing, was transformed into E.coli strain BL21.

Clone pMODC6-IL5/BL21 was grown over night in 5 ml LB containing 1 mg/LAmpicillin. 2 ml of this culture were diluted in 100 ml terrific broth(TB) containing 1 mg/L Ampicillin. The culture was induced by adding 0.1ml of a 1M solution of Ispropyl β-D-Thiogalactopyranoside (IPTG) whenthe culture reached an optical density OD600=0.7. 10 ml samples weretaken every 2 h. The samples were centrifugated 10 min at 4000×g. Thepellet was resuspended in 0.5 ml Lysis buffer containing 50 mM Tris-HCl,2 mM EDTA, 0.1% triton X-100 (pH8). After having added 20 μl of Lysozyme(40 mg/ml) and having incubated the tube 30 min at 4° C., the cells weresonicated for 2 min. 100 μl of a 50 mM MgCl₂ solution and 1 ml ofbenzonase were added. The cells were then incubated 30 min at roomtemperature and centrifugated 15 min at 13000×g.

The supernatant was discarded and the pellet was boiled 5 min at 98° C.in 100 μl of SDS loading buffer. 10 μl of the samples in loading bufferwere analyzed by SDS-PAGE under reducing conditions (FIG. 17 A). The gelof FIG. 17 A clearly demonstrates expression of the IL-5 construct. Thesamples loaded on the gel of FIG. 17 A were the following:

Lane M: Marker (NEB, Broad range prestained marker). Lane 1: cellextract of 1 ml culture before induction. Lane 2: cell extract of 1 mlculture 4 h after induction.

B. Cloning of IL-5 for Expression in Mammalian Cells (HEK-293T)

a) IL-5 Fused at its N-Terminus to an Amino Acid Linker Containing aCysteine Residue and Fused at its C-Terminus to the Fc Fragment

The template described under (A) (ATCC clone 37562) was used for thecloning of the following construct. The plasmid pMODB1-IL5 (a pETderivative) was digested with BamHI/XhoI to yield a small fragmentencoding IL5 fused to an N terminal amino acid linker containing acysteine. This fragment was ligated in the vector pCEP-SP-XA-Fc*(ΔXho)which had previously been digested with BamHI and XhoI. The ligation waselectroporated into E. coli strain TG1 and plasmid DNA of resultingclone pCEP-SP-IL5-Fc.2, whose sequence had been confirmed by DNAsequencing, was used to transfect HEK-293T cells. The resulting IL-5construct encoded by this plasmid had the amino acid sequenceADPGCGGGGGLA fused at the N-terminus of the IL-5 mature sequence. Thissequence comprises the amino acid linker sequence GCGGGGG containing acysteine and flanked by additional amino acids introduced during thecloning procedure. The IL-5 protein released by cleavage of the fusionprotein with Factor-Xa is named hereinafter “mouse C-IL-5-F” (SEQ IDNO:333).

After transfection and selection on Puromycin the culture supernatantwas analyzed by Western-Blot (FIG. 17 B) using an anti-His (mouse) andan anti-mouse IgG antibody conjugated to Horse radish peroxidase. Theanti-mouse IgG antibody conjugated to Horse radish peroxidase alsodetects Fc-fusion proteins. Purification of the protein was performed byaffinity chromatography on Protein-A resin. The result of FIG. 17 Bclearly demonstrates expression of the IL-5 construct.

The samples loaded on the Western-Blot of FIG. 17 B were the following:Lane 1: supernatant of HEK culture expressing IL5-Fc (20 μl). SDS-PAGEwas performed under reducing conditions. Lane 2: supernatant of HEKculture expressing IL13-Fc (20 μl). SDS-PAGE was performed under nonreducing conditions. Lane 3: supernatant of HEK culture expressingIL5-Fc (20 μl). SDS-PAGE was performed under non reducing conditions.

b) IL-5 Cloned with GST (Glutathion-S-Transferase) and an Amino AcidLinker Containing a Cysteine Residue Fused at its N-Terminus

IL-5 (ATCC 37562) was amplified with the primers Nhe-link1-IL13-F andIL5StopXho-R. After digestion with NheI and XhoI the insert was ligatedinto pCEP-SP-GST-EK which had been previously digested with NheI andXhoI. The resulting plasmid pCEP-SP-GST-IL5 was sequenced and used fortransfection of HEK-293T cells. The resulting IL-5 construct encoded bythis plasmid had the amino acid sequence LACGGGGG fused at theN-terminus of the IL-5 mature sequence. This sequence comprises theamino acid linker sequence ACGGGGG containing a cysteine residue andflanked by additional amino acids introduced during the cloningprocedure. The protein released by cleavage with enterokinase was namedhereinafter “mouse C-IL-5-S” (SEQ ID NO:334). The purification of theresulting protein was performed by affinity chromatography onGlutathione affinity resin.

C. Coupling of Mouse C-IL-5-F or Mouse C-IL-5-S to Qβ Capsid Protein

A solution of 120 μM Qβ capsid protein in 20 mM Hepes, 150 mM NaCl pH7.2 is reacted for 30 minutes with a 25 fold molar excess of SMPH(Pierce), diluted from a stock solution in DMSO, at 25° C. on a rockingshaker. The reaction solution is subsequently dialyzed twice for 2 hoursagainst 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzed Qβreaction mixture is then reacted with the mouse C-IL-5-F or mouseC-IL-5-S solution (end concentrations: 60 μM Qβ capsid protein, 60 μMmouse C-IL-5-F or mouse C-IL-5-S) for four hours at 25° C. on a rockingshaker. Coupling products are analysed by SDS-PAGE.

D. Coupling of Mouse Mouse C-IL-5-F or Mouse C-IL-5-S to Fr CapsidProtein

A solution of 120 μM fr capsid protein in 20 mM Hepes, 150 mM NaCl pH7.2 is reacted for 30 minutes with a 25 fold molar excess of SMPH(Pierce), diluted from a stock solution in DMSO, at 25° C. on a rockingshaker. The reaction solution is subsequently dialyzed twice for 2 hoursagainst 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzed frreaction mixture is then reacted with the mouse C-IL-5-F or mouseC-IL-5-S solution (end concentrations: 60 μM fr capsid protein, 60 μMmouse C-IL-5-F or mouse C-IL-5-S) for four hours at 25° C. on a rockingshaker. Coupling products are analysed by SDS-PAGE.

E. Coupling of Mouse C-IL-5-F or Mouse C-IL-5-S Solution toHBcAg-Lys-2Cys-Mut

A solution of 120 μM HBcAg-Lys-2cys-Mut capsid in 20 mM Hepes, 150 mMNaCl pH 7.2 is reacted for 30 minutes with a 25 fold molar excess ofSMPH (Pierce), diluted from a stock solution in DMSO, at 25° C. on arocking shaker. The reaction solution is subsequently dialyzed twice for2 hours against 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. Thedialyzed HBcAg-Lys-2cys-Mut reaction mixture is then reacted with themouse mouse C-IL-5-F or mouse C-IL-5-S solution (end concentrations: 60μM HBcAg-Lys-2cys-Mut, 60 μM mouse C-IL-5-F or mouse C-IL-5-S) for fourhours at 25° C. on a rocking shaker. Coupling products are analysed bySDS-PAGE.

F. Coupling of Mouse C-IL-5-F or Mouse C-IL-5-S Solution to Pili

A solution of 125 μM Type-1 pili of E. coli in 20 mM Hepes, pH 7.4, isreacted for 60 minutes with a 50-fold molar excess of cross-linker SMPH,diluted from a stock solution in DMSO, at RT on a rocking shaker. Thereaction mixture is desalted on a PD-10 column (Amersham-PharmaciaBiotech). The protein-containing fractions eluating from the column arepooled, and the desalted derivatized pili protein is reacted with themouse mouse C-IL-5-F or mouse C-IL-5-S solution (end concentrations: 60μM pili, 60 μM mouse C-IL-5-F or mouse C-IL-5-S) for four hours at 25°C. on a rocking shaker. Coupling products are analysed by SDS-PAGE.

Example 11 Introduction of an Amino Acid Linker Containing a CysteineResidue, Expression, Purification and Coupling of a Murine VascularEndothelial Growth Factor-2 (mVEGFR-2, FLK1) Fragment

A construct of the murine vascular endothelial growth factor-2(mVEGFR-2, FLK-1) comprising its second and third extracellular domainswas recombinantly expressed as a Fc-fusion protein with an amino acidlinker containing a cysteine residue at its C-terminus for coupling toVLPs and Pili. The protein sequences of the mVEGFR-2(2-3) was translatedfrom the cDNA sequences of mouse FLK-1 ((Matthews et al., Proc. Natl.Acad. Sci. USA 88: 9026-9030 (1991)): Accession no.: X59397; Ig-likeC2-type domain 2: amino acid 143-209; Ig-like C2-type domain 3: aminoacid 241-306). The mVEGFR-2 (2-3) construct comprises the sequence ofmVEGFR-2 from amino acid proline126 to lysine329 (in the numbering ofthe precursor protein). The construct also comprises, in addition to theImmunoglobulin-like C2-type domains 2 and 3, flanking regions precedingdomain 2 and following domain 3 in the sequence of mVEGFR-2, to addamino acid spacer moieties. An amino acid linker containing a cysteineresidue was fused to the C-terminus of the mVEGFR-2 sequence throughcloning into pCEP-SP-EK-Fc* vector (EXAMPLE 1). The fragment of mVEGFR-2cloned into pCEP-SP-EK-Fc* vector encoded the following amino acidsequence (SEQ ID NO:345):

PFIAS VSDQHGIVYI TENKNKTVVI PCRGSISNLNVSLCARYPEK RFVPDGNRIS WDSEIGFTLP SYMISYAGMVFCEAKINDET YQSIMYIVVV VGYRIYDVIL SPPHEIELSAGEKLVLNCTA RTELNVGLDF TWHSPPSKSH HKKIVNRDVKPFPGTVAKMF LSTLTIESVT KSDQGEYTCV ASSGRMIKRN RTFVRVHTKP

Expression of Recombinant mVEGFR-2(2-3) in Eukaryotic Cells

Recombinant mVEGFR-2(2-3) was expressed in EBNA 293 cells using thepCEP-SP-EK-Fc* vector. The pCEP-SP-EK-Fc* vector has a BamHI and an NheIsites, encodes an amino acid linker containing one cysteine residue, anenterokinase cleavage site, and C-terminally a human Fc region. ThemVEGFR-2(2-3) was amplified by PCR with the primer pair BamH1-FLK1-F andNheI-FLK1-B from a mouse 7-day embryo cDNA (Marathon-Ready cDNA,Clontech). For the PCR reaction, 10 pmol of each oligo and 0.5 ng of thecDNA (mouse 7-day embryo cDNA Marathon-Ready cDNA, Clontech) was used inthe 50 μl reaction mixture (1 l of Advantage 2 polymerase mix (50×), 0.2mM dNTPs and 5 l 10× cDNA PCR reaction buffer). The temperature cycleswere as follows: 5 cycles a 94 C for 1 minute, 94 C for 30 seconds, 54 Cfor 30 seconds, 72 C for 1 minute followed by 5 cycles of 94 C (30seconds), 54 C (30 seconds), 70 C (1 minute) and followed by 30 cycles94 C (20 seconds), 54 C (30 seconds) and 68 C (1 minute). The PCRproduct was digested with BamH1 and NheI and inserted into thepCEP-SP-EK-Fc* vector digested with the same enzymes. Resulting plasmidwas named mVEGFR-2(2-3)-pCep-EK-Fc. All other steps were performed bystandard molecular biology protocols.

Oligos:

1. Primer BamHI-FLK1-F (SEQ ID NO: 343) 5′-CGCGGATCCATTCATCGCCTCTGTC-3′2. Primer NheI-FLK1-B (SEQ ID NO: 344) 5′-CTAGCTAGCTTTGTGTGAACTCGGAC-3′

Transfection and Expression of Recombinant mVEGFR-2(2-3)

EBNA 293 cells were transfected with the mVEGFR-2(2-3)-pCep-Ek-Fcconstruct described above and serum free supernatant of cells washarvested for purification as described in EXAMPLE 1.

Purification of Recombinant mVEGFR-2(2-3)

Protein A purification of the expressed Fc-EK-mVEGFR-2(2-3) proteins wasperformed as described in EXAMPLE 1. Subsequently, after binding of thefusion protein to Protein A, mVEGFR-2(2-3) was cleaved from the Fcportion bound to protein A using enterokinase (EnterokinaseMax,Invitrogen). Digestion was conducted over night at 37 C (2.5 unitsenterokinase/100 μl Protein A beads with bound fusion protein). Thereleased VEGFR-2(2-3) was separated from the Fc-portion still bound toprotein A beads by short centrifugation in chromatography columns (MicroBio Spin, Biorad). In order to remove the enterokinase the flow throughwas treated with enterokinase away (Invitrogen) according to theinstructions of the manufacturer.

Example 12 Coupling of Murine VEGFR-2 Peptide to Qβ Capsid Protein,HbcAg-Lys-2Cys-Mut and Pili and Immunization of Mice with VLP-Peptideand Pili-Peptide Vaccines A. Coupling of Murine VEGFR-2 Peptides to VLPsand Pili

The following peptides was chemically synthesized (by Eurogentec,Belgium): murine VEGFR-2 peptide CTARTELNVGLDFTWHSPPSKSHHKK and used forchemical coupling to Pili as described below.

Coupling of Murine VEGFR-2 Peptides to Pili:

A solution of 1400 μl of 1 mg/ml pili protein in 20 mM Hepes, pH 7.4,was reacted for 60 minutes with 85 μl of a 100 mM Sulfo-MBS (Pierce)solution in (H₂O) at RT on a rocking shaker. The reaction mixture wasdesalted on a PD-10 column (Amersham-Pharmacia Biotech), Theprotein-containing fractions eluting from the column were pooled(containing approximately 1.4 mg protein) and reacted with a 2.5-foldmolar excess (final volume) of murine VEGFR-2 peptide respectively. Forexample, to 200 μl eluate containing approximately 0.2 mg derivatizedpili, 2.4 μl of a 10 mM peptide solution (in DMSO) were added. Themixture was incubated for four hours at 25° C. on a rocking shaker andsubsequently dialyzed against 2 liters of 20 mM Hepes, pH 7.2 overnightat 4° C. Coupling results were analyzed by SDS-PAGE under reducingconditions and are shown in FIG. 18 A. Supernatant (S) and pellet (P) ofeach sample were loaded on the gel, as well pili and pili derivatizedwith Sulfo-MBS cross-linker (Pierce). The samples loaded on the gel ofFIG. 18 A were the following:

Lane 1: Marker proteins; lane 2-5: coupled samples (Pili murine: Pilicoupled to murine peptide; Pili human: Pili coupled to human peptide);lane 6: pili derivatized with Sulfo-MBS cross-linker; lane 7-9: threefractions of the eluate of the PD-10 column. Fraction 2 is the peakfraction, fraction 1 and 3 are fractions taken at the border of thepeak. Coupling bands were clearly visible on the gel, demonstrating thesuccessful coupling of murine VEGFR-2 to pili.

Coupling of Murine VEGFR-2 Peptide to Qβ Capsid Protein:

A solution of 1 ml of 1 mg/ml Qβ capsid protein in 20 mM Hepes, 150 mMNaCl pH 7.4 was reacted for 45 minutes with 20 μl of 100 mM Sulfo-MBS(Pierce) solution in (H₂O) at RT on a rocking shaker. The reactionsolution was subsequently dialyzed twice for 2 hours against 2 L of 20mM Hepes, pH 7.4 at 4° C. 1000 μl of the dialyzed reaction mixture wasthen reacted with 12 μl of a 10 mM peptide solution (in DMSO) for fourhours at 25° C. on a rocking shaker. The reaction mixture wassubsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, pH 7.4at 4° C. Coupling results were analyzed by SDS-PAGE under reducingconditions and are shown in FIG. 18 B. Supernatant (S) of each samplewas loaded on the gel, as well as Qβ capsid protein and Qβ capsidprotein derivatized with Sulfo-MBS cross-linker. Coupling was done induplicate. The following samples were loaded on the gel:

Lane 1: Marker proteins; lane 2, 5: Qβ capsid protein; lane 3, 6 Qβcapsid protein derivatized with Sulfo-MBS; lane 4, 7: Qβ capsid proteincoupled to murine VEGFR-2 peptide. Coupling bands were clearly visibleon the gel, demonstrating the successful coupling of murine VEGFR-2 toQβ capsid protein.

Coupling of Murine VEGFR-2 Peptide to HbcAg-Lys-2Cys-Mut:

A solution of 3 ml of 0.9 mg/ml cys-free HbcAg capsid protein (EXAMPLE31) in PBS, pH 7.4 was reacted for 45 minutes with 37.5 μl of a 100 mMSulfo-MBS (Pierce) solution in (H₂O) at RT on a rocking shaker. Thereaction solution was subsequently dialyzed overnight against 2 L of 20mM Hepes, pH 7.4. After buffer exchange the reaction solution wasdialyzed for another 2 hours against the same buffer. The dialyzedreaction mixture was then reacted with 3 μl of a 10 mM peptide solution(in DMSO) for 4 hours at 25° C. on a rocking shaker. The reactionmixture was subsequently dialyzed against 2 liters of 20 mM Hepes, pH7.4 overnight at 4° C. followed by buffer exchange and another 2 hoursof dialysis against the same buffer. Coupling results were analyzed bySDS-PAGE under reducing conditions and are shown in FIG. 18 C. Thesupernatant (S) of each sample was loaded on the gel, as well asHbcAg-lys-2cys-Mut protein and HbcAg-lys-2cys-Mut protein derivatizedwith Sulfo-MBS cross-linker. Coupling was done in duplicate. Couplingreactions were conducted in a 2.5 fold and 10 fold molar excess ofpeptide. The following samples were loaded on the gel:

Lane 1: Marker proteins; lane 2, 4, 6, 8: Supernatant (S) and pellet (P)of coupling reactions performed with 10 fold molar excess of peptide;lane 3, 5, 7, 9: Supernatant (S) and pellet (P) of coupling reactionsperformed with 2.5 fold molar excess of peptide; lane 10:HbcAg-lys-2cys-Mut derivatized with Sulfo-MBS; lane 11:HbcAg-lys-2cys-Mut.Coupling bands were clearly visible on the gel, demonstrating thesuccessful coupling of murine VEGFR-2 to HbcAg-lys-2cys-Mut protein.

B. Immunization of Mice:

Pili-Peptide Vaccine:

Female C3H-HeJ (Toll-like receptor 4 deficient) and C3H-HeN (wild-type)mice were vaccinated with the murine VEGFR-2 peptide coupled to piliprotein without the addition of adjuvants. Approximately 100 μg of totalprotein of each sample was diluted in PBS to 200 μl and injectedsubcutaneously on day 0, day 14 and day 28. Mice were bledretroorbitally on day 14, 28 and day 42 and serum of day 42 was analyzedusing a human VEGFR-2 specific ELISA.

Qβ Capsid Protein-Peptide Vaccine:

Female Black 6 mice were vaccinated with the murine VEGFR-2 peptidecoupled to Qβ capsid protein with and without the addition of adjuvant(Aluminiumhydroxid). Approximately 100 μg of total protein of eachsample was diluted in PBS to 200 μl and injected subcutaneously on day0, day 14 and day 28. Mice were bled retroorbitally on day 14, 28 andday 42 and serum of day 42 was analyzed using a human VEGFR-2 specificELISA.

HbcAg-Lys-2Cys-Mut Vaccines:

Female Black 6 mice were vaccinated with the murine VEGFR-2 peptidecoupled to HbcAg-lys-2cys-Mut protein with and without the addition ofadjuvant (Aluminiumhydroxid). Approximately 100 μg of total protein ofeach sample was diluted in PBS to 200 μl and injected subcutaneously onday 0, day 14 and day 28. Mice were bled retroorbitally on day 14, 28and day 42 and serum of day 42 was analyzed using a human VEGFR-2specific ELISA.

C. ELISA

Sera of immunized mice were tested in ELISA with immobilized murineVEGFR-2 peptide. Murine VEGFR-2 peptide was coupled to bovine RNAse Ausing the chemical cross-linker Sulfo-SPDP. ELISA plates were coatedwith coupled RNAse A at a concentration of 10 μg/ml. The plates wereblocked and then incubated with serially diluted mouse sera. Boundantibodies were detected with enzymatically labeled anti-mouse IgGantibody. As a control, preimmune sera of the same mice were alsotested. Control ELISA experiments using sera from mice immunized withuncoupled carrier showed that the antibodies detected were specific forthe respective peptide. The results are shown in FIG. 4-6.

Pili-Peptide Vaccine:

The result of the ELISA is shown in FIG. 18 D. Results for indicatedserum dilutions are shown as optical density at 450 nm. The average ofthree mice each (including standard deviations) are shown. Allvaccinated mice made IgG antibody titers against the murine VEGFR-2peptide. No difference was noted between mice deficient for theToll-like receptor 4 and wild-type mice, demonstrating theimmunogenicity of the self-antigen murine VEGFR-2 peptide, when coupledto pili, in mice. The vaccines injected in the mice are designating thecorresponding analyzed sera.

Qβ Capsid Protein-Peptide Vaccine:

Results for indicated serum dilutions are shown in FIG. 18 E as opticaldensity at 450 nm. The average of two mice each (including standarddeviations) are shown. All vaccinated mice made IgG antibody titersagainst the murine VEGFR-2 peptide, demonstrating the immunogenicity ofthe self-antigen murine VEGFR-2 peptide, when coupled to Qβ capsidprotein, in mice. The vaccines injected in the mice are designating thecorresponding analyzed sera.

HbcAg-Lvs-2Cys-Mut Vaccine:

Results for indicated serum dilutions are shown in FIG. 18 F as opticaldensity at 450 nm. The average of three mice each (including standarddeviations) are shown.

All vaccinated mice made IgG antibody titers against the murine VEGFR-2peptide, demonstrating the immunogenicity of the self-antigen murineVEGFR-2 peptide, when coupled to Qβ capsid protein, in mice. Thevaccines injected in the mice are designating the corresponding analyzedsera.

Example 13 Coupling of Aβ 1-15 Peptides to HBc-Ag-Lys-2Cys-Mut and FrCapsid Protein

The following Aβ peptide was chemically synthesized(DAEFRHDSGYEVHHQGGC), a peptide which comprises the amino acid sequencefrom residue 1-15 of human Aβ, fused at its C-terminus to the sequenceGGC for coupling to VLPs and Pili.

A. a.) Coupling of Aβ 1-15 Peptide to HBc-Ag-Lys-2Cys-Mut Using theCross-Linker SMPH.

A solution of 833.3 μl of 1.2 mg/ml HBc-Ag-lys-2cys-Mut protein in 20 mMHepes 150 mM NaCl pH 7.4 was reacted for 30 minutes with 17 μl of asolution of 65 mM SMPH (Pierce) in H₂O, at 25° C. on a rocking shaker.The reaction solution was subsequently dialyzed twice for 2 hoursagainst 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. in a dialysistubing with Molecular Weight cutoff 10000 Da. 833.3 μl of the dialyzedreaction mixture was then reacted with 7.1 μl of a 50 mM peptide stocksolution (peptide stock solution in DMSO) for two hours at 15° C. on arocking shaker. The reaction mixture was subsequently dialyzed overnightagainst 1 liters of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. The samplewas then frozen in aliquots in liquid Nitrogen and stored at −80° C.until immunization of the mice.

b) Coupling of Aβ 1-15 Peptide to Fr Capsid Protein Using theCross-Linker SMPH.

A solution of 500 μl of 2 mg/ml fr capsid protein in 20 mM Hepes 150 mMNaCl pH 7.4 was reacted for 30 minutes with 23 μl of a solution of 65 mMSMPH (Pierce) in H₂O, at 25° C. on a rocking shaker. The reactionsolution was subsequently dialyzed twice for 2 hours against 1 L of 20mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. in a dialysis tubing withMolecular Weight cutoff 10000 Da. 500 μl of the dialyzed reactionmixture was then reacted with 5.7 μl of a 50 mM peptide stock solution(peptide stock solution in DMSO) for two hours at 15° C. on a rockingshaker. The reaction mixture was subsequently dialyzed overnight against1 liter of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. The sample was thenfrozen in aliquots in liquid Nitrogen and stored at −80° C. untilimmunization of the mice. Samples of the coupling reaction were analyzedby SDS-PAGE under reducing conditions.

The results of the coupling experiments were analyzed by SDS-PAGE, andare shown in FIG. 19 A. Clear coupling bands corresponding to thecoupling of Aβ 1-15 either to fr capsid protein or toHBc-Ag-lys-2cys-Mut were visible on the gel, and are indicated by arrowsin the figure, demonstrating successful coupling of Aβ 1-15 to fr capsidprotein and to HBc-Ag-lys-2cys-Mut capsid protein. Multiple couplingbands were visible for the coupling to fr capsid protein, while mainlyone coupling band was visible for HBc-Ag-lys-2cys-Mut.

The following samples were loaded on the gel of FIG. 19 A.

1: Protein Marker (kDa Marker 7708S BioLabs. Molecular weight markerbands from the top of the gel: 175, 83, 62, 47.5, 32.5, 25, 16.5, 6.5kDa). 2: derivatized HBc-Ag-lys-2cys-Mut. 3: HBc-Ag-lys-2cys-Mut coupledwith Aβ1-15, supernatant of the sample taken at the end of the couplingreaction, and centrifuged. 4: HBc-Ag-lys-2cys-Mut coupled with Aβ1-15,pellet of the sample taken at the end of the coupling reaction, andcentrifuged. 5: derivatized fr capsid protein. 6: fr capsid proteincoupled with Aβ1-15, supernatant of the sample taken at the end of thecoupling reaction, and centrifuged. 4: fr capsid protein coupled withAβ1-15, pellet of the sample taken at the end of the coupling reaction,and centrifuged.

B. Immunization of Balb/c Mice

Female Balb/c mice were vaccinated twice on day 0 and day 14subcutaneously with either 10 μg of fr capsid protein coupled to Aβ 1-15(Fr-Aβ 1-15) or 10 μg of HBc-Ag-lys-2cys-Mut coupled to Aβ 1-15(HBc-Aβ1-15) diluted in sterile PBS. Mice were bled retroorbitally onday 22 and sera were analysed in an Aβ-1-15-specific ELISA.

C. ELISA

The Aβ 1-15 peptide was coupled to bovine RNAse A using the chemicalcross-linker sulfo-SPDP. ELISA plates were coated with Aβ 1-15-RNAseconjugate at a concentration of 10 μg/ml. The plates were blocked andthen incubated with serially diluted serum samples. Bound antibodieswere detected with enzymatically labeled anti-mouse IgG. As a control,serum from a naive mouse was also tested.

Shown on FIG. 19 B are the ELISA signals obtained on day 22 with thesera of the mice immunized with vaccines Fr-Aβ 1-15, and HBc-Aβ1-15respectively. A control serum from a naïve mouse (preimmune serum) wasalso included. Results from different serum dilutions are shown asoptical density at 450 nm. Average results from three vaccinated miceeach are shown. All vaccinated mice had Aβ 1-15-specific IgG antibodiesin their serum.

Example 14 Coupling of Aβ 1-15, Aβ 1-27 and Aβ 33-42 Peptides to Type IPili

Coupling of Aβ 1-15, Aβ 1-27 and Aβ 33-42 Peptides to Pili Using theCross-Linker SMPH.

The following Aβ peptides were chemically synthesized:DAEFRHDSGYEVHHQGGC (“Aβ 1-15”), a peptide which comprises the amino acidsequence from residue 1-15 of human Aβ, fused at its C-terminus to thesequence GGC for coupling to Pili and VLPs,DAEFRHDSGYEVHHQKLVFFAEDVGSNGGC (“Aβ 1-27”) a peptide which comprises theamino acid sequence from residue 1-27 of human Aβ, fused at itsC-terminus to the sequence GGC for coupling to Pili and VLPs, andCGHGNKSGLMVGGVVIA (“Aβ33-42”) a peptide which comprises the amino acidsequence from residue 33-42 of Aβ, fused at its N-terminus to thesequence CGHGNKS for coupling to Pili and VLPs. All three peptides wereused for chemical coupling to Pili as described in the following.

A solution of 2 ml of 2 mg/ml Pili in 20 mM Hepes 150 mM NaCl pH 7.4 wasreacted for 45 minutes with 468 μl of a solution of 33.3 mM SMPH(Pierce) in H₂O, at 25° C. on a rocking shaker. The reaction solutionwas loaded on a PD 10 column (Pharmacia) and eluted with 6×500 μl of 20mM Hepes 150 mM NaCl pH 7.4. Fractions were analyzed by dotting on aNitrocellulose (Schleicher & Schuell) and stained with Amidoblack.Fractions 3-6 were pooled. The samples were then frozen in aliquots inliquid Nitrogen and stored at −80° C. until coupling.

200 μl of the thawed desalted reaction mixture was then mixed with 200μl DMSO and 2.5 μl of each of the corresponding 50 mM peptide stocksolutions in DMSO, for 3.5 hours at RT on a rocking shaker. 400 μl ofthe reaction mixture was subsequently dialyzed three times for one houragainst 1 liter of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. in adialysis tubing with Molecular Weight cutoff 10000 Da. The samples werethen frozen in aliquots in liquid Nitrogen and stored at −80° C.

Sample preparation for SDS-Page was performed as follows: 100 μl of thedialyzed coupling reaction was incubated for 10 minutes in 10% TCA onice and subsequently centrifuged. The pellet was resuspended in 50 μl8.5 M Guanidine-HCl solution and incubated for 15 minutes at 70° C. Thesamples were then precipitated with ethanol, and after a secondcentrifugation step, the pellet was resuspended in sample buffer.

The results of the coupling experiments were analyzed by SDS-PAGE underreducing conditions. Clear coupling bands were visible for all threepeptides, demonstrating coupling of Aβ peptides to Pili.

Example 15 Vaccination of APP23 Mice with Aβ Peptides Coupled to QβCapsid Protein

A. Immunization of APP23 Mice

Three different Aβ peptides (Aβ 1-27-Gly-Gly-Cys-NH2;H-Cys-Gly-His-Gly-Asn-Lys-Ser-Aβ 33-42; Aβ 1-15-Gly-Gly-Cys-NH2) werecoupled to Qβ capsid protein. The resulting vaccines were termed “Qb-Ab1-15”, “Qb-Ab 1-27” and “Qb-Ab 33-42”. 8 months old female APP23 micewhich carry a human APP transgene (Sturchler-Pierrat et al., Proc. Natl.Acad. Sci. USA 94: 13287-13292 (1997)) were used for vaccination. Themice were injected subcutaneously with 25 μg vaccine diluted in sterilePBS and 14 days later boosted with the same amount of vaccine. Mice werebled from the tail vein before the start of immunization and 7 daysafter the booster injection. The sera were analyzed by ELISA.

B. ELISA

Aβ 1-40 and Aβ 1-42 peptide stocks were made in DMSO and diluted incoating buffer before use. ELISA plates were coated with 0.1 μg/well Aβ1-40 or Aβ 1-42 peptide. The plates were blocked and then incubated withserially diluted mouse serum. Bound antibodies were detected withenzymatically labeled anti-mouse IgG antibody. As a control, seraobtained before vaccination were also included. The serum dilutionshowing a mean three standard deviations above baseline was calculatedand defined as “ELISA titer”. All three vaccines tested were immunogenicin APP23 mice and induced high antibody titers against the Aβ peptides1-40 and/or Aβ 1-42. The results are shown in FIG. 20. No specificantibodies were detected in preimmune sera of the same mice (not shown).

Shown on FIG. 20 are the ELISA signals obtained on day 22 with the seraof the mice immunized with vaccines Fr-Aβ 1-15, and HBc-Aβ1-15respectively. A control serum from a naïve mouse (preimmune serum) wasalso included. Results from different serum dilutions are shown asoptical density at 450 nm. Average results from three vaccinated miceeach are shown.

Mice A21-A30 received the vaccine Qb-Ab 1-15, mice A31-A40 receivedQb-Ab 1-27 and mice A41-49 received Qb-Ab 33-42. For each mouse, Aβ 1-40and Aβ 1-42 peptide-specific serum antibody titers were determined onday 21 by ELISA. The ELISA titers defined as the serum dilution showinga mean three standard deviations above baseline are shown for individualmice. Mice vaccinated with Qb-Ab 1-15 or Qb-Ab 1-27 made high antibodytiters against both Aβ 1-40 and Aβ 1-42 whereas mice vaccinated withQb-Ab 33-42 had only high antibody titers against the Aβ 1-42 peptide.

Example 16 Coupling of Fab Antibody Fragments to Qβ Capsid Protein

A solution of 4.0 mg/ml Qβ capsid protein in 20 mM Hepes, 150 mM NaCl pH7.2 was reacted for 30 minutes with a 2.8 mM SMPH (Pierce) (from a stocksolution dissolved in DMSO) at 25° C. on a rocking shaker. The reactionsolution was subsequently dialyzed twice for 2 hours against 2 l of 20mM Hepes, 150 mM NaCl, pH 7.2 at 4° C.

The Fab fragment of human IgG, produced by papain digestion of humanIgG, was purchased from Jackson Immunolab. This solution (11.1 mg/ml)was diluted to a concentration of 2.5 mg/ml in 20 mM Hepes, 150 mM NaClpH 7.2 and allowed to react with different concentrations (0-1000 μM) ofeither dithiothreitol (DTT) or tricarboxyethylphosphine (TCEP) for 30minutes at 25° C.

Coupling was induced by mixing the derivatized and dialysed Qβ capsidprotein solution with non-reduced or reduced Fab solution (finalconcentrations: 1.14 mg/ml Qβ and 1.78 mg/ml Fab) and proceededovernight at 25° C. on a rocking shaker.

The reaction products were analysed on 16% SDS-PAGE gels under reducingconditions. Gels were stained with Coomassie Brilliant Blue. The resultsare shown in FIG. 21.

A coupling product of about 40 kDa could be detected in samples in whichthe Fab had been reduced before coupling by 25-1000 μM TCEP and 25-100μM DTT (FIG. 21, arrow), but not at 10 μM TCEP, 10 μM DTT or 1000 μMDTT. The coupled band also reacted with an anti-Qβ antiserum (data notshown) clearly demonstrating the covalent coupling of the Fab fragmentto Qβ capsid protein.The samples loaded on the gel of FIG. 21 were the following:

Lane 1: Molecular weight marker. Lane 2 and 3: derivatized Qβ capsidprotein before coupling. Lane 4-13: Qβ-Fab coupling reactions afterreduction of Fab with 4: Qβ-Fab coupling reactions after reduction ofFab with 10 μM TCEP. 5: Qβ-Fab coupling reactions after reduction of Fabwith 25 μM TCEP. 6: Qβ-Fab coupling reactions after reduction of Fabwith 50 μM TCEP, 7: Qβ-Fab coupling reactions after reduction of Fabwith 100 μM TCEP. 8: Qβ-Fab coupling reactions after reduction of Fabwith 1000 μM TCEP. 9: Qβ-Fab coupling reactions after reduction of Fabwith 10 μM DTT. 10: Qβ-Fab coupling reactions after reduction of Fabwith 25 μM DTT. 11: Qβ-Fab coupling reactions after reduction of Fabwith 50 μM DTT. 12: Qβ-Fab coupling reactions after reduction of Fabwith 100 μM DTT. 13: Qβ-Fab coupling reactions after reduction of Fabwith 1000 μM DTT. Lane 14: Fab before coupling. The gel was stained withCoomassie Brilliant Blue. Molecular weights of marker proteins are givenon the left margin. The arrow indicates the coupled band.

Example 17 Vaccination of APP23 mice with Aβ peptides coupled to Qβcapsid protein

A. Immunization of APP23 Mice

Three different Aβ peptides (Aβ 1-27-Gly-Gly-Cys-NH2;H-Cys-Gly-His-Gly-Asn-Lys-Ser-Aβ 33-42; Aβ 1-15-Gly-Gly-Cys-NH2) werecoupled to Qβ capsid protein. The resulting vaccines were termed “Qb-Ab1-15”, “Qb-Ab 1-27” and “Qb-Ab 33-42”. 8 months old female APP23 micewhich carry a human APP transgene (Sturchler-Pierrat et al., Proc. Natl.Acad. Sci. USA 94: 13287-13292 (1997)) were used for vaccination. Themice were injected subcutaneously with 25 μg vaccine diluted in sterilePBS and 14 days later boosted with the same amount of vaccine. Mice werebled from the tail vein before the start of immunization and 7 daysafter the booster injection. The sera were analyzed by ELISA.

B. ELISA

Aβ 1-40 and Aβ 1-42 peptide stocks were made in DMSO and diluted incoating buffer before use. ELISA plates were coated with 0.1 μg/well Aβ1-40 or Aβ 1-42 peptide. The plates were blocked and then incubated withserially diluted mouse serum. Bound antibodies were detected withenzymatically labeled anti-mouse IgG antibody. As a control, seraobtained before vaccination were also included. The serum dilutionshowing a mean three standard deviations above baseline was calculatedand defined as “ELISA titer”. All three vaccines tested were immunogenicin APP23 mice and induced high antibody titers against the Aβ peptides1-40 and/or Aβ 1-42. The results are shown in FIG. 20. No specificantibodies were detected in preimmune sera of the same mice (not shown).

Shown on FIG. 20 are the ELISA signals obtained on day 22 with the seraof the mice immunized with vaccines Qb-Ab 1-15, Qb-Ab 1-27 and Qb-Ab33-42, respectively. Mice A21-A30 received the vaccine Qb-Ab 1-15, miceA31-A40 received Qb-Ab 1-27 and mice A41-49 received Qb-Ab 33-42. Foreach mouse, Aβ 1-40 and Aβ 1-42 peptide-specific serum antibody titerswere determined on day 21 by ELISA. The ELISA titers defined as theserum dilution showing a mean three standard deviations above baselineare shown for individual mice. Mice vaccinated with Qb-Ab 1-15 or Qb-Ab1-27 made high antibody titers against both Aβ 1-40 and Aβ 1-42 whereasmice vaccinated with Qb-Ab 33-42 had only high antibody titers againstthe Aβ 1-42 peptide. The very strong immune responses obtained with thehuman Aβ peptides in the transgenic mice expressing human Aβ transgene,demonstrate that by coupling Aβ peptides to Qβ capsid protein, tolerancetowards the self-antigen can be overcome.

Example 18 Construction, Expression and Purification of Mutant Qρ CoatProteins

Construction of pQβ-240

The plasmid pQβ10 (Kozlovska, T M, et al., Gene 137:133-137) was used asan initial plasmid for the construction of pQβ-240. The mutationLys13→Arg was created by inverse PCR. The inverse primers were designedin inverted tail-to-tail directions:

5′-GGTAACATCGGTCGAGATGGAAAACAAACTCTGGTCC-3′ and5′-GGACCAGAGTTTGTTTTCCATCTCGACCGATGTTACC-3′.

The products of the first PCR were used as templates for the second PCRreaction, in which an upstream primer

5′-AGCTCGCCCGGGGATCCTCTAG-3′ and a downstream primer

5′-CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3′ were used. The product ofthe second PCR was digested with XbaI and Mph1103I and cloned into thepQβ10 expression vector, which was cleaved by the same restrictionenzymes. The PCR reactions were performed with PCR kit reagents andaccording to producer protocol (MBI Fermentas, Vilnius, Lithuania).

Sequencing using the direct label incorporation method verified thedesired mutations. E. coli cells harbouring pQβ-240 supported efficientsynthesis of 14-kD protein co migrating upon PAGE with control Qβ coatprotein isolated from Qβ phage particles.

Resulting amino acid sequence: (SEQ ID NO: 255)AKLETVTLGNIGRDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQKYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

Construction of pQβ-243

The plasmid pQβ10 was used as an initial plasmid for the construction ofpQβ-243. The mutation Asn10→Lys was created by inverse PCR. The inverseprimers were designed in inverted tail-to-tail directions:

5'-GGCAAAATTAGAGACTGTTACTTTAGGTAAGATCGG-3' and5'-CCGATCTTACCTAAAGTAACAGTCTCTAATTTTGCC-3'.

The products of the first PCR were used as templates for the second PCRreaction, in which an upstream primer

5′-AGCTCGCCCGGGGATCCTCTAG-3′ and a downstream primer

5′-CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3′ were used. The product ofthe second PCR was digested with XbaI and Mph1103I and cloned into thepQβ10 expression vector, which was cleaved by the same restrictionenzymes. The PCR reactions were performed with PCR kit reagents andaccording to producer protocol (MBI Fermentas, Vilnius, Lithuania).

Sequencing using the direct label incorporation method verified thedesired mutations. E. coli cells harbouring pQβ-243 supported efficientsynthesis of 14-kD protein co migrating upon PAGE with control Qβ coatprotein isolated from Qβ phage particles.

Resulting amino acid sequence: (SEQ ID NO: 256)AKLETVTLGKIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQKYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

Construction of pQβ-250

The plasmid pQβ-240 was used as an initial plasmid for the constructionof pQβ-250. The mutation Lys2→Arg was created by site-directedmutagenesis. An upstream primer

5′-GGCCATGGCACGACTCGAGACTGTTACTTTAGG-3′ and a downstream primer5′-GATTTAGGTGACACTATAG-3′ were used for the synthesis of the mutantPCR-fragment, which was introduced into the pQβ-185 expression vector atthe unique restriction sites NcoI and HindIII. The PCR reactions wereperformed with PCR kit reagents and according to producer protocol (MBIFermentas, Vilnius, Lithuania).

Sequencing using the direct label incorporation method verified thedesired mutations. E. coli cells harbouring pQβ-250 supported efficientsynthesis of 14-kD protein co migrating upon PAGE with control Qβ coatprotein isolated from Qβ phage particles.

Resulting amino acid sequence: (SEQ ID NO: 257)ARLETVTLGNIGRDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQKYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

Construction of pQβ-251

The plasmid pQβ10 was used as an initial plasmid for the construction ofpQβ-251. The mutation Lys16→Arg was created by inverse PCR. The inverseprimers were designed in inverted tail-to-tail directions:

5′-GATGGACGTCAAACTCTGGTCCTCAATCCGCGTGGGG -3′ and5′-CCCCACGCGGATTGAGGACCAGAGTTTGACGTCCATC -3′.

The products of the first PCR were used as templates for the second PCRreaction, in which an upstream primer

5′-AGCTCGCCCGGGGATCCTCTAG-3′ and a downstream primer

5′-CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3′ were used. The product ofthe second PCR was digested with XbaI and Mph1103I and cloned into thepQβ10 expression vector, which was cleaved by the same restrictionenzymes. The PCR reactions were performed with PCR kit reagents andaccording to producer protocol (MBI Fermentas, Vilnius, Lithuania).

Sequencing using the direct label incorporation method verified thedesired mutations. E. coli cells harbouring pQβ-251 supported efficientsynthesis of 14-kD protein co migrating upon PAGE with control Qβ coatprotein isolated from Qβ phage particles. The resulting amino acidsequence encoded by this construct is shown in SEQ. ID NO: 259.

Construction of pQβ-259

The plasmid pQβ-251 was used as an initial plasmid for the constructionof pQβ-259. The mutation Lys2→Arg was created by site-directedmutagenesis. An upstream primer

5′-GGCCATGGCACGACTCGAGACTGTTACTTTAGG-3′ and a downstream primer5′-GATTTAGGTGACACTATAG-3′ were used for the synthesis of the mutantPCR-fragment, which was introduced into the pQβ-185 expression vector atthe unique restriction sites NcoI and HindIII. The PCR reactions wereperformed with PCR kit reagents and according to producer protocol (MBIFermentas, Vilnius, Lithuania).

Sequencing using the direct label incorporation method verified thedesired mutations. E. coli cells harbouring pQβ-259 supported efficientsynthesis of 14-kD protein co migrating upon PAGE with control Qβ coatprotein isolated from Qβ phage particles.

Resulting amino acid sequence: (SEQ ID NO: 258)AKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQKYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

General procedures for Expression and purification of Qβ and Qβ MutantsExpression

Transform E. coli JM109 with Q-beta expression plasmids. Inoculate 5 mlof LB liquid medium with 20 μg/ml ampicillin with clones transformedwith Q-beta expression plasmids. Incubate at 37° C. for 16-24 h withoutshaking.

Inoculate 100-300 ml of LB medium, containing 20 μg/ml, 1:100 with theprepared inoculum. Incubate at 37° C. overnight without shaking.Inoculate M9+1% Casamino acids+0.2% glucose medium in flasks with theprepared inoculum 1:50, incubate at 37° C. overnight under shaking.

Purification

Solutions and Buffers for the Purification Procedure:

1. Lysis Buffer LB

-   -   50 mM Tris-HCl pH8.0 with 5 mM EDTA, 0.1% tritonX100 and fresh!        prepared PMSF till 5 micrograms per ml. Without lysozyme and        DNAse.

2. SAS

-   -   Saturated ammonium sulphate in water

3. Buffer NET.

-   -   20 mM Tris-HCl, pH 7.8 with 5 mM EDTA and    -   150 mM NaCl.

4. PEG

-   -   40% (w/v) polyethylenglycol 6000 in NET

Disruption and Lyses

Frozen cells were resuspended in LB at 2 ml/g cells. The mixture wassonicated with 22 kH five times for 15 seconds, with intervals of 1 minto cool the solution on ice. The lysate was then centrifuged at 14 000rpm, for 1 h using a Janecki K 60 rotor. The centrifugation stepsdescribed below were all performed using the same rotor, exceptotherwise stated. The supernatant was stored at 4° C., while cell debriswere washed twice with LB. After centrifugation, the supernatants of thelysate and wash fractions were pooled.

Fractionation

A saturated ammonium sulphate solution was added dropwise under stirringto the above pooled lysate. The volume of the SAS was adjusted to be onefifth of total volume, to obtain 20% of saturation. The solution wasleft standing overnight, and was centrifuged the next day at 14 000 rpm,for 20 min. The pellet was washed with a small amount of 20% ammoniumsulphate, and centrifuged again. The obtained supernatants were pooled,and SAS was added dropwise to obtain 40% of saturation. The solution wasleft standing overnight, and was centrifuged the next day at 14 000 rpm,for 20 min. The obtained pellet was solubilised in NET buffer.

Chromatography

The capsid protein resolubilized in NET buffer was loaded on a SepharoseCL-4B column. Three peaks eluted during chromatography. The first onemainly contained membranes and membrane fragments, and was notcollected. Capsids were contained in the second peak, while the thirdone contained other E. coli proteins.

The peak fractions were pooled, and the NaCl concentration was adjustedto a final concentration of 0.65 M. A volume of PEG solutioncorresponding to one half of the pooled peak fraction was added dropwiseunder stirring. The solution was left to stand overnight withoutstirring. The capsid protein was sedimented by centrifugation at 14 000rpm for 20 min. It was then solubilized in a minimal volume of NET andloaded again on the Sepharose CL-4B column. The peak fractions werepooled, and precipitated with ammonium sulphate at 60% of saturation(w/v). After centrifugation and resolubilization in NET buffer, capsidprotein was loaded on a Sepharose CL-6B column for rechromatography.

Dialysis and Drying

The peak fractions obtained above were pooled and extensively dialysedagainst sterile water, and lyophilized for storage.

Expression and purification Qβ-240

Cells (E. coli JM 109, transformed with the plasmid pQβ-240) wereresuspended in LB, sonicated five times for 15 seconds (water icejacket) and centrifuged at 13000 rpm for one hour. The supernatant wasstored at 4° C. until further processing, while the debris were washed 2times with 9 ml of LB, and finally with 9 ml of 0.7 M urea in LB. Allsupernatants were pooled, and loaded on the Sepharose CL-4B column. Thepooled peak fractions were precipitated with ammonium sulphate andcentrifuged. The resolubilized protein was then purified further on aSepharose 2B column and finally on a Sepharose 6B column. The capsidpeak was finally extensively dialyzed against water and lyophilized asdescribed above. The assembly of the coat protein into a capsid wasconfirmed by electron microscopy.

Expression and Purification Qβ-243

Cells (E. coli RR1) were resuspended in LB and processed as described inthe general procedure. The protein was purified by two successive gelfiltration steps on the sepharose CL-4B column and finally on asepharose CL-2B column. Peak fractions were pooled and lyophilized asdescribed above. The assembly of the coat protein into a capsid wasconfirmed by electron microscopy.

Expression and Purification of Qβ-250

Cells (E. coli JM 109, transformed with pQβ-250) were resuspended in LBand processed as described above. The protein was purified by gelfiltration on a Sepharose CL-4B and finally on a Sepharose CL-2B column,and lyophilized as described above. The assembly of the coat proteininto a capsid was confirmed by electron microscopy.

Expression and Purification of Qβ-259

Cells (E. coli JM 109, transformed with pQβ-259) were resuspended in LBand sonicated. The debris were washed once with 10 ml of LB and a secondtime with 10 ml of 0.7 M urea in LB. The protein was purified by twogel-filtration chromatogaphy steps, on a Sepharose CL-4 B column. Theprotein was dialyzed and lyophilized, as described above. The assemblyof the coat protein into a capsid was confirmed by electron microscopy.

Example 19 Desensitization of Allergic Mice with PLA2 Coupled to QβCapsid Protein

C. Desensitization of Allergic Mice by Vaccination

Female CBA/J mice (8 weeks old) were sensitized with PLA2: Per mouse,0.1 ug PLA2 from Latoxan (France) was adsorbed to 1 mg Alum (Imject,Pierce) in a total volume of 66 ul by vortexing for 30 min and theninjected subcutaneously. This procedure was repeated every 14 days for atotal of four times. This treatment led to the development ofPLA2-specific serum IgE but no IgG2a antibodies. 1 month after the lastsensitization, mice were injected subcutaneously with 10 ug vaccineconsisting of recombinant PLA2 coupled to Qβ capsid protein. One and 2weeks later they were again treated with the same amount of vaccine. Oneweek after the last treatment, mice were bled and then challengedintraperitoneally with 25 μg PLA2 (Latoxan) and rectal temperature wasmeasured for 60 min using a calibrated digital thermometer. As a controlsensitized mice which had not been treated with Qβ capsid protein-PLA2were used. Whereas all control mice experienced an anaphylactic responsereflected in a dramatic drop in rectal temperature after PLA2 challenge,vaccinated mice were fully or at least partially protected. Results areshown in FIG. 25 A.

B. ELISA

ELISA plates (Maxisorp, Nunc) were coated with PLA2 (Latoxan) at 5μg/ml. The plates were blocked and then incubated with serially dilutedserum. For the detection of IgE antibodies, serum was pretreated withprotein G beads (Pharmacia) for 60 min on a shaker at room temperature.The beads were removed by centrifugation and the supernatant was usedfor ELISA. Antibodies bound to PLA2 were detected with enzymaticallylabeled anti-mouse IgG2a or IgE antibodies. ELISA titers were determinedat half maximal optical density (OD50%) and expressed as −log 5 of100-fold prediltued sera for IgG2a and as −log 5 of 10-fold predilutedsera for IgE. For all mice, PLA2-specific IgG2a and IgE in serum weredetermined before and at the end of the vaccine treatment. Vaccinationled to a dramatic increase of PLA2-specific IgG2a whereas no consistentchanges in IgE titers were noted. These results indicate that thevaccination led to an induction of a Th1-like immune response (reflectedby the production of IgG2a). Results are shown in FIG. 25 B.

The Anaphylactic response in vaccinated and non-vaccinated mice is shownin FIG. 25A.

Mice were sensitized to PLA2 and then treated 3× subcutaneously with 10μg vaccine consisting of PLA2 coupled to Qβ capsid protein. Control micewere sensitized but not vaccinated. One week after the last vaccinationall mice were challenged intraperitoneally with 25 μg PLA2 and theanaphylactic response was monitored by measuring the rectal temperaturefor 60 min. Whereas all control mice showed a dramatic drop in bodytemperature, vaccinated mice were fully or at least partially protectedfrom an anaphylactic reaction.

The induction of PLA2-specific IgG2a by vaccination is shown in FIG. 25B.

Mice were sensitized to PLA2 and then treated 3× with 10 ug vaccineconsisting of PLA2 coupled to Qβ capsid protein. Control mice weresensitized but not vaccinated. Serum was taken from sensitized micebefore the start of the treatment and after completion of treatment,before challenge. In vaccinated mice (left hand of panel) a dramaticincrease of PLA2-specific IgG2a was observed.

Example 20 Expression, Refolding, Purification and Coupling of Pla₂-Cys(Also Called PLA₂ Fusion Protein)

Expression and Preparation of Inclusion Bodies

The pET11a Plasmid containing the PLA₂-Cys gene of example xxx wastransformed into E. coli BL21DE3Ril1 (Stratagene). An overnight culturewas grown in dYT medium containing 100 μg/ml Ampicillin and 15 μg/mlChloramphenicol. The culture was diluted in fresh dYT medium containingAmpicillin and Chloramphenicol, and grown at 37° C. until OD_(600 nm)=1was reached. The culture was induced with 1 mM IPTG, and grown foranother 4 hours. Cells were collected by centrifugation, and resuspendedin PBS buffer containing 0.5 mg/ml Lysozyme. After incubation on ice,cells were sonicated on ice, and MgCl₂ added to a concentration of 10mM. 6 μl of Benzonase (Merck) were added to the cell lysate, and thelysate was incubated 30 minutes at RT. Triton was added to a finalconcentration of 1%, and the lysate was further incubated for 30 minuteson ice. The inclusion body (IB) pellet was collected by centrifugationfor 10 minutes at 13000 g. The inclusion body pellet was washed in washbuffer containing 20 mM Tris, 23% sucrose, 1 mM EDTA, pH 8.0. The IBswere solubilized in 6 M Guanidinium-HCl, 20 mM Tris, pH 8.0, containing200 mM DTT. The solubilized IBs were centrifuged at 50000 g and thesupernatant dialyzed against 6 M Guanidinium-HCl, 20 mM Tris, pH 8.0 andsubsequently against the same buffer containing 0.1 mM DTT. Oxidizedglutathion was added to a final concentration of 50 mM, and thesolubilized IBs were incubated for 1 h. at RT. The solubilized IBs weredialyzed against 6 M Guanidinium-HCL, 20 mM Tris, pH 8.0. Theconcentration of the IB solution was estimated by Bradford analysis andSDS-PAGE.

B. Refolding and Purification

The IB solution was added slowly in three portions, every 24 h., to afinal concentration of 3 μM, to the refolding buffer containing 2 mMEDTA, 0.2 mM Benzamidin, 0.2 mM 6 aminocapronic acid, 0.2 mMGuanidinium-HCl, 0.4 M L-Arginin, pH 6.8, to which 5 mM reducedGlutathion and 0.5 mM oxidized Glutathion were added prior to initiationof refolding at 4° C. The refolding solution was concentrated to onehalf of its volume by Ultrafiltration using a YM10 membrane (Millipore)and dialyzed against PBS, pH 7.2, containing 0.1 mM DTT. The protein wasfurther concentrated by ultrafiltration and loaded onto a Superdex G-75column (Pharmacia) equilibrated in 20 mM Hepes, 150 mM NaCl, 0.1 mM DTT,4° C. for purification. The pH of the equilibration buffer was adjustedto 7.2 at RT. The monomeric fractions were pooled.

C. Coupling

A solution of 1.5 mg Qβ in 0.75 mL 20 mM Hepes, 150 mM NaCl, pH 7.4

was reacted with 0.06 mL Sulfo-SMPB (Pierce; 31 mM Stock in H₂O) for 45min. at RT. The reaction mixture was dialyzed overnight against 20 mMHepes, 150 mM NaCl, pH 7.4 and 0.75 mL of this solution were mixed with1.5 mL of a PLA₂-Cys solution in 0.1 mM DTT (62 μM) and 0.43 mL of 20 mMHepes, 150 mM NaCl, 137 μM DTT, pH 7.4 adjusted at RT. The couplingreaction was left to proceed for 4 h. at RT, and the reaction mixturewas dialyzed overnight against 20 mM Hepes, 150 mM NaCl, pH 7.4 usingSpectra Por dialysis tubing, MW cutoff 300 000 Da (Spectrum). Thecoupling reaction was analyzed by SDS-PAGE and coomassie staining, andWestern blotting, using either a rabbit anti-bee venom antiserum(diluted 1:10000), developed with a goat anti-rabbit alkalinephosphatase conjugate (diluted 1:10000), or a rabbit anti-Qβ antiserum(1:5000), developed with a goat anti-rabbit alkaline phosphataseconjugate (diluted 1:10000). Samples were run in both cases underreducing conditions.

The result of the coupling reaction is shown in FIG. 26. Bandscorresponding to the coupling product of Qβ capsid protein to PLA₂-Cysare clearly visible in the coomassie stained SDS-PAGE (left panel), theanti-Qβ Western Blot (center panel) and the anti-PLA2 Western blot(right panel) of the coupling reactions between Qβ capsid protein andPLA₂-Cys, and are indicated by an arrow in the figure. 15 μl of thecoupling reactions and 50 μl of the dialyzed coupling reactions wereloaded on the gel.

Lane 1: Protein marker. 2: Dialyzed coupling reaction 1. 3: Couplingreaction 1. 4: Coupling reaction 2. 5: coupling reaction 2. 6: Couplingreaction 1. 7: Dialyzed coupling reaction 1. 8: Protein Marker. 9:Coupling reaction 2. 10: Coupling reaction 1. 11: Dialyzed couplingreaction 1. 12: Protein Marker.

Example 21 Coupling of Anti-Idiotypic IgE Mimobody VAE051 to Qβ,Immunization of Mice and Testing of Antisera

A solution of 4.0 mg/ml Qβ capsid protein in 20 mM Hepes, 150 mM NaCl pH7.2 was reacted for 30 minutes with 10 fold molar excess SMPH (Pierce)(from a 100 mM stock solution dissolved in DMSO) at 25° C. on a rockingshaker. The reaction solution was subsequently dialyzed twice for 2hours against 2 l of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. TheVAE051 solution (2.4 mg/ml) was reducted with an equimolar concentrationof TCEP for 60 min at 25° C.

46 μl of the dialyzed Qβ reaction mixture was then reacted with 340 μlof the TCEP-treated VAE051 solution (2.4 mg/ml) in a total volume of 680μl of 50 mM sodium acetate buffer at

16° C. for 2 h on a rocking shaker.

The reaction products were analysed on 16% SDS-PAGE gels under reducingconditions. Gels were either stained with Coomassie Brilliant Blue. Thetwo additional band in the coupling reactions (which are absent in VAEor Qβ solutions) represent the heavy chain and the light chain of theVAE051 coupled to Qβ (FIG. 28 A). Identity of the bands were confirmedby Western blotting with antibodies specific for heavy and light chains,respectively.

Immunization of Mice

The Qβ-VAE051 coupling solution was dialysed against 20 mM Hepes, 150 mMNaCl, pH 7.2 using a membrane with a cut-off of 300000 Da. 50 μg of theQβ-VAE051 were injected intraperitoneal in two female Balb/c mice at day0 and day 14. Mice were bled retroorbitally on day 28 and their serumwas analyzed using IgE- and VAE051-specific ELISAs.

ELISA

ELISA plates were coated with human IgE at a concentration of 0.8 μg/mlor with 10 μg/ml VAE051. The plates were blocked and then incubated withserially diluted mouse sera. Bound antibodies were detected withenzymatically labeled anti-mouse IgG antibody (FIG. 28 B).

Both mice showed high reactivity to VAE051 as well as the human IgE.Preimmune sera of the same mice did not show any reactivity againstVAE051 and IgE (FIG. 28 B). This demonstrates that antibodies againstthe anti-idiotypic IgE mimobody VAE051 have been produced which alsorecognize the “parent” molecule IgE.

Example 22 High Occupancy Coupling of DerpI Peptide to Wt Qβ CapsidProtein Using the Cross-Linker SMPH

The Derp 1,2 peptide, to which a cysteine was added N-terminally forcoupling, was chemically synthesized and had the following sequence:H2N-CQIYPPNANKIREALAQTHSA-COOH. This peptide was used for chemicalcoupling to wt Qβ capsid protein and as described in the following.

D. Coupling of Flag Peptide to Qβ Capsid Protein

Qβ capsid protein in 20 mM Hepes, 150 mM NaCl, pH 7.2, at aconcentration of 2 mg/ml, was reacted with a 5- or 20-fold excess of thecross-linker SMPH (Pierce) for 30 mM. at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. The dialyzed reactionmixture was then reacted with a 5-fold excess of Derp 1,2 peptide fortwo hours at 25° C. on a rocking shaker.

The result of the coupling reaction can be seen on FIG. 24.

Coupling bands corresponding to 1, 2 and 3 peptides per subunit,respectively, are clearly visible on the gel, and are indicated byarrows. An average of two peptides per subunit were displayed on thecapsid.

The samples loaded on the gel of FIG. 24 were the following:

Lane 1: Protein Marker. 2: Qβ capsid protein derivatized with a 5-foldexcess of SMPH. 3: Qβ capsid protein derivatized with a 20-fold excessof SMPH. 4: Coupling reaction of 5-fold derivatized Qβ capsid protein.5: Coupling reaction of 20-fold derivatized Qβ capsid protein.

Example 23 Insertion of a Peptide Containing a Lysine Residue into thec/e1 Epitope of HBcAg(1-149)

The c/e1 epitope (residues 72 to 88) of HBcAg is located in the tipregion on the surface of the Hepatitis B virus capsid (HBcAg). A part ofthis region (Proline 79 and Alanine 80) was genetically replaced by thepeptide Gly-Gly-Lys-Gly-Gly (HBcAg-Lys construct). The introduced Lysineresidue contains a reactive amino group in its side chain that can beused for intermolecular chemical crosslinking of HBcAg particles withany antigen containing a free cysteine group.

HBcAg-Lys DNA, having the amino acid sequence shown in SEQ ID NO:158,was generated by PCRs: The two fragments encoding HBcAg fragments (aminoacid residues 1 to 78 and 81 to 149) were amplified separately by PCR.The primers used for these PCRs also introduced a DNA sequence encodingthe Gly-Gly-Lys-Gly-Gly peptide. The HBcAg (1 to 78) fragment wasamplified from pEco63 using primers EcoRIHBcAg(s) and Lys-HBcAg(as). TheHBcAg (81 to 149) fragment was amplified from pEco63 using primersLys-HBcAg(s) and HBcAg(1-149)Hind(as). Primers Lys-HBcAg(as) andLys-HBcAg(s) introduced complementary DNA sequences at the ends of thetwo PCR products allowing fusion of the two PCR products in a subsequentassembly PCR. The assembled fragments were amplified by PCR usingprimers EcoRIHBcAg(s) and HbcAg(1-149)Hind(as).

For the PCRs, 100 pmol of each oligo and 50 ng of the template DNAs wereused in the 50 ml reaction mixtures with 2 units of Pwo polymerase, 0.1mM dNTPs and 2 mM MgSO4. For both reactions, temperature cycling wascarried out as follows: 94° C. for 2 minutes; 30 cycles of 94° C. (1minute), 50° C. (1 minute), 72° C. (2 minutes).

Primer Sequences:

EcoRIHBcAg(s): (SEQ ID NO: 79) (5′-CCGGAATTCATGGACATTGACCCTTATAAAG-3′);Lys-HBcAg(as): (SEQ ID NO: 80) (5′-CCTAGAGCCACCTTTGCCACCATCTTCTAAATTAGTACCCACCCAG GTAGC-3′); Lys-HBcAg(s):(SEQ ID NO: 81) (5′- GAAGATGGTGGCAAAGGTGGCTCTAGGGACCTAGTAGTCAGTTATGTC -3′); HBcAg(1-149)Hind(as): (SEQ ID NO: 82)(5′-CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGAAG-3′).

For fusion of the two PCR fragments by PCR 100 pmol of primersEcoRIHBcAg(s) and HBcAg(1-149)Hind(as) were used with 100 ng of the twopurified PCR fragments in a 50 ml reaction mixture containing 2 units ofPwo polymerase, 0.1 mM dNTPs and 2 mM MgSO₄. PCR cycling conditionswere: 94° C. for 2 minutes; 30 cycles of 94° C. (1 minute), 50° C. (1minute), 72° C. (2 minutes). The assembled PCR product was analyzed byagarose gel electrophoresis, purified and digested for 19 hours in anappropriate buffer with EcoRI and HindIII restriction enzymes. Thedigested DNA fragment was ligated into EcoRI/HindIII-digested pKK vectorto generate pKK-HBcAg-Lys expression vector. Insertion of the PCRproduct into the vector was analyzed by EcoRI/HindIII restrictionanalysis and DNA sequencing of the insert.

Example 24 Expression and Partial Purification of HBcAg-Lys

E. coli strain XL-1 blue was transformed with pKK-HBcAg-Lys. 1 ml of anovernight culture of bacteria was used to innoculate 100 ml of LB mediumcontaining 100 μg/ml ampicillin. This culture was grown for 4 hours at37° C. until an OD at 600 nm of approximately 0.8 was reached. Inductionof the synthesis of HBcAg-Lys was performed by addition of IPTG to afinal concentration of 1 mM. After induction, bacteria were furthershaken at 37° C. for 16 hours. Bacteria were harvested by centrifugationat 5000×g for 15 minutes. The pellet was frozen at -20° C. The pelletwas thawed and resuspended in bacteria lysis buffer (10 mM Na₂HPO₄, pH7.0, 30 mM NaCl, 0.25% Tween-20, 10 mM EDTA, 10 mM DTT) supplementedwith 200 μg/ml lysozyme and 10 μl of Benzonase (Merck). Cells wereincubated for 30 minutes at room temperature and disrupted using aFrench pressure cell. Triton X-100 was added to the lysate to a finalconcentration of 0.2%, and the lysate was incubated for 30 minutes onice and shaken occasionally. E. coli cells harboring pKK-HBcAg-Lysexpression plasmid or a control plasmid were used for induction ofHBcAg-Lys expression with IPTG. Prior to the addition of IPTG, a samplewas removed from the bacteria culture carrying the pKK-HBcAg-Lys plasmidand from a culture carrying the control plasmid. Sixteen hours afteraddition of IPTG, samples were again removed from the culture containingpKK-HBcAg-Lys and from the control culture. Protein expression wasmonitored by SDS-PAGE followed by Coomassie staining

The lysate was then centrifuged for 30 minutes at 12,000×g in order toremove insoluble cell debris. The supernatant and the pellet wereanalyzed by Western blotting using a monoclonal antibody against HBcAg(YVS1841, purchased from Accurate Chemical and Scientific Corp.,Westbury, N.Y., USA), indicating that a significant amount of HBcAg-Lysprotein was soluble. Briefly, lysates from E. coli cells expressingHBcAg-Lys and from control cells were centrifuged at 14,000×g for 30minutes. Supernatant (=soluble fraction) and pellet (=insolublefraction) were separated and diluted with SDS sample buffer to equalvolumes. Samples were analyzed by SDS-PAGE followed by Western blottingwith anti-HBcAg monoclonal antibody YVS 1841.

The cleared cell lysate was used for step-gradient centrifugation usinga sucrose step gradient consisting of a 4 ml 65% sucrose solutionoverlaid with 3 ml 15% sucrose solution followed by 4 ml of bacteriallysate. The sample was centrifuged for 3 hrs with 100,000×g at 4° C.After centrifugation, 1 ml fractions from the top of the gradient werecollected and analyzed by SDS-PAGE followed by Coomassie staining. TheHBcAg-Lys protein was detected by Coomassie staining.

The HBcAg-Lys protein was enriched at the interface between 15 and 65%sucrose indicating that it had formed a capsid particle. Most of thebacterial proteins remained in the sucrose-free upper layer of thegradient, therefore step-gradient centrifugation of the HBcAg-Lysparticles led both to enrichment and to a partial purification of theparticles.

Example 25 Chemical Coupling of FLAG Peptide to HbcAg-Lys Using theHeterobifunctional Cross-Linker SPDP

Synthetic FLAG peptide with a Cysteine residue at its amino terminus(amino acid sequence CGGDYKDDDDK (SEQ ID NO:147)) was coupled chemicallyto purified HBcAg-Lys particles in order to elicit an immune responseagainst the FLAG peptide. 600 ml of a 95% pure solution of HBcAg-Lysparticles (2 mg/ml) were incubated for 30 minutes at room temperaturewith the heterobifunctional cross-linker N-Succinimidyl3-(2-pyridyldithio) propionate (SPDP) (0.5 mM). After completion of thereaction, the mixture was dialyzed overnight against 1 liter of 50 mMPhosphate buffer (pH 7.2) with 150 mM NaCl to remove free SPDP. Then 500ml of derivatized HBcAg-Lys capsid (2 mg/ml) were mixed with 0.1 mM FLAGpeptide (containing an amino-terminal cysteine) in the presence of 10 mMEDTA to prevent metal-catalyzed sulfhydryl oxidation. The reaction wasmonitored through the increase of the optical density of the solution at343 nm due to the release of pyridine-2-thione from SPDP upon reactionwith the free cysteine of the peptide. The reaction of derivatized Lysresidues with the peptide was complete after approximately 30 minutes.

The FLAG decorated particles were injected into mice.

Example 26 Construction of pMPSV-gp140cys

The gp140 gene was amplified by PCR from pCytTSgp140FOS using oligosgp140CysEcoRI and SalIgp140. For the PCRs, 100 pmol of each oligo and 50ng of the template DNAs were used in the 50 ml reaction mixtures with 2units of Pwo polymerase, 0.1 mM dNTPs and 2 mM MgSO4. For bothreactions, temperature cycling was carried out as follows: 94° C. for 2minutes; 30 cycles of 94° C. (0.5 minutes), 55° C. (0.5 minutes), 72° C.(2 minutes).

The PCR product was purified using QiaEXII kit, digested with SalI/EcoRIand ligated into vector pMPSVHE cleaved with the same enzymes.

Oligo Sequences:

Gp140CysEcoRI: (SEQ ID NO: 83)5′-GCCGAATTCCTAGCAGCTAGCACCGAATTTATCTAA-3′; SalIgp140: (SEQ ID NO: 84)5′- GGTTAAGTCGACATGAGAGTGAAGGAGAAATAT-3′.

Example 27 Expression of pMPSVgp140Cys

pMPSV140Cys (20 μg) was linearized by restriction digestion. Thereaction was stopped by phenol/chloroform extraction, followed by anisopropanol precipitation of the linearized DNA. The restrictiondigestion was evaluated by agarose gel electrophoresis. For thetransfection, 5.4 μg of linearized pMPSVgp140-Cys was mixed with 0.6 μgof linearized pSV2Neo in 30 μl H₂O and 30 μl of 1 M CaCl₂ solution wasadded. After addition of 60 μl phosphate buffer (50 mM HEPES, 280 mMNaCl, 1.5 mM Na_(z) HPO₄, pH 7.05), the solution was vortexed for 5seconds, followed by an incubation at room temperature for 25 seconds.The solution was immediately added to 2 ml HP-1 medium containing 2% FCS(2% FCS medium). The medium of an 80% confluent BHK21 cell culture(6-well plate) was then replaced by the DNA containing medium. After anincubation for 5 hours at 37° C. in a CO₂ incubator, the DNA containingmedium was removed and replaced by 2 ml of 15% glycerol in 2% FCSmedium. The glycerol containing medium was removed after a 30 secondincubation phase, and the cells were washed by rinsing with 5 ml of HP-1medium containing 10% FCS. Finally 2 ml of fresh HP-1 medium containing10% FCS was added.

Stably transfected cells were selected and grown in selection medium(HP-1 medium supplemented with G418) at 37° C. in a CO₂ incubator. Whenthe mixed population was grown to confluency, the culture was split totwo dishes, followed by a 12 h growth period at 37° C. One dish of thecells was shifted to 30° C. to induce the expression of solubleGP140-FOS. The other dish was kept at 37° C.

The expression of soluble GP140-Cys was determined by Western blotanalysis. Culture media (0.5 ml) was methanol/chloroform precipitated,and the pellet was resuspended in SDS-PAGE sample buffer. Samples wereheated for 5 minutes at 95° C. before being applied to a 15% acrylamidegel. After SDS-PAGE, proteins were transferred to Protan nitrocellulosemembranes (Schleicher & Schuell, Germany) as described by Bass and Yang,in Creighton, T. E., ed., Protein Function: A Practical Approach, 2ndEdn., IRL Press, Oxford (1997), pp. 29-55. The membrane was blocked with1% bovine albumin (Sigma) in TBS (10×TBS per liter: 87.7 g NaCl, 66.1 gTrizma hydrochloride (Sigma) and 9.7 g Trizma base (Sigma), pH 7.4) for1 hour at room temperature, followed by an incubation with an anti-GP140or GP-160 antibody for 1 hour. The blot was washed 3 times for 10minutes with TBS-T (TBS with 0.05% Tween20), and incubated for 1 hourwith an alkaline-phosphatase-anti-mouse/rabbit/monkey/human IgGconjugate. After washing 2 times for 10 minutes with TBS-T and 2 timesfor 10 minutes with TBS, the development reaction was carried out usingalkaline phosphatase detection reagents (10 ml AP buffer (100 mMTris/HCl, 100 mM NaCl, pH 9.5) with 50 μl NBT solution (7.7% Nitro BlueTetrazolium (Sigma) in 70% dimethylformamide) and 37 μl of X-Phosphatesolution (5% of 5-bromo-4-chloro-3-indolyl phosphate indimethylformamide).

Example 28 Purification of gp140Cys

An anti-gp120 antibody was covalently coupled to a NHS/EDC activateddextran and packed into a chromatography column. The supernatant,containing GP140Cys is loaded onto the column and after sufficientwashing, GP140Cys was eluted using 0.1 M HCl. The eluate was directlyneutralized during collection using 1 M Tris pH 7.2 in the collectiontubes.

Disulfide bond formation might occur during purification, therefore thecollected sample is treated with 10 mM DTT in 10 mM Tris pH 7.5 for 2hours at 25° C.

DTT is remove by subsequent dialysis against 10 mM Mes; 80 mM NaCl pH6.0. Finally GP140Cys is mixed with alphavirus particles containing theJUN residue in E2 as described in Example 16.

Example 29 Construction of PLA₂-Cys

The PLA₂ gene was amplified by PCR from pAV3PLAfos using oligos EcoRIPLAand PLA-Cys-hind. For the PCRs, 100 pmol of each oligo and 50 ng of thetemplate DNAs were used in the 50 ml reaction mixtures with 2 units ofPwo polymerase, 0.1 mM dNTPs and 2 mM MgSO₄. For the reaction,temperature cycling was carried out as follows: 94° C. for 2 minutes; 30cycles of 94° C. (0 5 minutes), 55° C. (0.5 minutes), 72° C. (2minutes).

The PCR product was purified using QiaEXII kit, digested withEcoRI/HindIII and ligated into vector pAV3 cleaved with the sameenzymes.

Oligos

EcoRIPLA: (SEQ ID NO: 85) 5′-TAACCGAATTCAGGAGGTAAAAAGATATGG-3′PLA Cys-hind: (SEQ ID NO: 86) 5′-GAAGTAAAGCTTTTAACCACCGCAACCACCAGAAG-3′.

Example 30 Expression and Purification of PLA₂-Cys

For cytoplasmic production of Cys tagged proteins, E. coli XL-1-Bluestrain was transformed with the vectors pAV3::PLA and pPLA-Cys. Theculture was incubated in rich medium in the presence of ampicillin at37° C. with shaking. At an optical density (550 nm) of, 1 mM IPTG wasadded and incubation was continued for another 5 hours. The cells wereharvested by centrifugation, resuspended in an appropriate buffer (e.g.,Tris-HCl, pH 7.2, 150 mM NaCl) containing DNase, RNase and lysozyme, anddisrupted by passage through a french pressure cell. Aftercentrifugation (Sorvall RC-5C, SS34 rotor, 15000 rpm, 10 min, 4° C.),the pellet was resuspended in 25 ml inclusion body wash buffer (20 mMtris-HCl, 23% sucrose, 0.5% Triton X-100, 1 mM EDTA, pH8) at 4° C. andrecentrifuged as described above. This procedure was repeated until thesupernatant after centrifugation was essentially clear. Inclusion bodieswere resuspended in 20 ml solubilization buffer (5.5 M guanidiniumhydrochloride, 25 mM tris-HCl, pH 7.5) at room temperature and insolublematerial was removed by centrifugation and subsequent passage of thesupernatant through a sterile filter (0.45 nm). The protein solution waskept at 4° C. for at least 10 hours in the presence of 10 mM EDTA and100 mM DTT and then dialyzed three times against 10 volumes of 5.5 Mguanidinium hydrochloride, 25 mM tris-HCl, 10 mM EDTA, pH 6. Thesolution was dialyzed twice against 51 2 M urea, 4 mM EDTA, 0.1 M NH₄Cl,20 mM sodium borate (pH 8.3) in the presence of an appropriate redoxshuffle (oxidized glutathione/reduced glutathione; cystine/cysteine).The refolded protein was then applied to an ion exchange chromatography.The protein was stored in an appropriate buffer with a pH above 7 in thepresence of 2-10 mM DTT to keep the cysteine residues in a reduced form.Prior to coupling of the protein with the alphavirus particles, DTT wasremoved by passage of the protein solution through a Sephadex G-25 gelfiltration column.

Example 31 Construction of a HBcAg Devoid of Free Cysteine Residues andContaining an Inserted Lysine Residue

A Hepatitis core Antigen (HBcAg), referred to herein asHBcAg-lys-2cys-Mut, devoid of cysteine residues at positionscorresponding to 48 and 107 in SEQ ID NO:134 and containing an insertedlysine residue was constructed using the following methods.

The two mutations were introduced by first separately amplifying threefragments of the HBcAg-Lys gene prepared as described above in Example23 with the following PCR primer combinations. PCR methods essentiallyas described in Example 1 and conventional cloning techniques were usedto prepare the HBcAg-lys-2cys-Mut gene.

In brief, the following primers were used to prepare fragment 1:

Primer 1: EcoRIHBcAg(s) (SEQ ID NO: 148) CCGGAATTCATGGACATTGACCCTTATAAAGPrimer 2: 48as (SEQ ID NO: 149) GTGCAGTATGGTGAGGTGAGGAATGCTCAGGAGACTC

The following primers were used to prepare fragment 2:

Primer 3: 48s (SEQ ID NO: 150) GSGTCTCCTGAGCATTCCTCACCTCACCATACTGCACPrimer 4: 107as (SEQ ID NO: 151) CTTCCAAAAGTGAGGGAAGAAATGTGAAACCAC

The following primers were used to prepare fragment 3:

Primer 5: HBcAg149hind-as (SEQ ID NO: 152)CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGAAGCGTTGATAG Primer 6: 107s(SEQ ID NO: 153) GTGGTTTCACATTTCTTCCCTCACTTTTGGAAG

Fragments 1 and 2 were then combined with PCR primers EcoRIHBcAg(s) and107 as to give fragment 4. Fragment 4 and fragment 3 were then combinedwith primers EcoRIHBcAg(s) and HBcAg149hind-as to produce the fulllength gene. The full length gene was then digested with the EcoRI(GAATTC) and HindIII (AAGCTT) enzymes and cloned into the pKK vector(Pharmacia) cut at the same restriction sites.

Example 32 Blockage of Free Cysteine Residues of a HBcAg Followed byCross-Linking

The free cysteine residues of the HBcAg-Lys prepared as described abovein Example 23 were blocked using Iodacetamide. The blocked HBcAg-Lys wasthen cross-linked to the FLAG peptide with the hetero-bifunctionalcross-linker m-maleimidonbenzoyl-N-hydroxysuccinimide ester (Sulfo-MBS).

The methods used to block the free cysteine residues and cross-link theHBcAg-Lys are as follows. HBcAg-Lys (550 μg/ml) was reacted for 15minutes at room temperature with Iodacetamide (Fluka Chemie, Brugg,Switzerland) at a concentration of 50 mM in phosphate buffered saline(PBS) (50 mM sodium phosphate, 150 mM sodium chloride), pH 7.2, in atotal volume of 1 ml. The so modified HBcAg-Lys was then reactedimmediately with Sulfo-MBS (Pierce) at a concentration of 330 μMdirectly in the reaction mixture of step 1 for 1 hour at roomtemperature. The reaction mixture was then cooled on ice, and dialyzedagainst 1000 volumes of PBS pH 7.2. The dialyzed reaction mixture wasfinally reacted with 300 μM of the FLAG peptide (CGGDYKDDDDK (SEQ IDNO:147)) containing an N-terminal free cysteine for coupling to theactivated HBcAg-Lys, and loaded on SDS-PAGE for analysis.

The resulting patterns of bands on the SDS-PAGE gel showed a clearadditional band migrating slower than the control HBcAg-Lys derivatizedwith the cross-linker, but not reacted with the FLAG peptide. Reactionsdone under the same conditions without prior derivatization of thecysteines with Iodacetamide led to complete cross-linking of monomers ofthe HBcAg-Lys to higher molecular weight species.

Example 33 Isolation and Chemical Coupling of FLAG Peptide to Type-1Pili of Escherichia coli Using a Heterobifunctional Cross-Linker

A. Introduction

Bacterial pili or fimbriae are filamentous surface organelles producedby a wide range of bacteria. These organelles mediate the attachment ofbacteria to surface receptors of host cells and are required for theestablishment of many bacterial infections like cystitis,pyelonephritis, new born meningitis and diarrhea.

Pili can be divided in different classes with respect to their receptorspecificity (agglutination of blood cells from different species), theirassembly pathway (extracellular nucleation, general secretion,chaperone/usher, alternate chaperone) and their morphological properties(thick, rigid pili; thin, flexible pili; atypical structures includingcapsule; curli; etc). Examples of thick, rigid pili forming a righthanded helix that are assembled via the so called chaperone/usherpathway and mediate adhesion to host glycoproteins include Type-1 pili,P-pili, S-pili, F1C-pili, and 987P-pili). The most prominent and bestcharacterized members of this class of pili are P-pili and Type-1 pili(for reviews on adhesive structures, their assembly and the associateddiseases see Soto, G. E. & Hultgren, S. J., J. Bacteriol. 181:1059-1071(1999); Bullitt & Makowski, Biophys. J. 74:623-632 (1998); Hung, D. L. &Hultgren, S. J., J. Struct, Biol. 124:201-220 (1998)).

Type-1 pili are long, filamentous polymeric protein structures on thesurface of E. coli. They possess adhesive properties that allow forbinding to mannose-containing receptors present on the surface ofcertain host tissues. Type-1 pili can be expressed by 70-80% of all E.coli isolates and a single E. coli cell can bear up to 500 pili.Type-pili reach a length of typically 0.2 to 2 μM with an average numberof 1000 protein subunits that associate to a right-handed helix with3.125 subunits per turn with a diameter of 6 to 7 nm and a central holeof 2.0 to 2.5 nm.

The main Type-1 pilus component, FimA, which represents 98% of the totalpilus protein, is a 15.8 kDa protein. The minor pilus components FimF,FimG and FimH are incorporated at the tip and in regular distances alongthe pilus shaft (Klemm, P. & Krogfelt, K. A., “Type I fimbriae ofEscherichia coli,” in: Fimbriae. Klemm, P. (ed.), CRC Press Inc., (1994)pp. 9-26). FimH, a 29.1 kDa protein, was shown to be the mannose-bindingadhesin of Type-1 pili (Krogfelt, K. A., et al., Infect. Immun.58:1995-1998 (1990); Klemm, P., et al., Mol. Microbiol. 4:553-560(1990); Hanson, M. S. & Brinton, C. C. J., Nature 17:265-268 (1988)),and its incorporation is probably facilitated by FimG and FimF (Klemm,P. & Christiansen, G., Mol. Gen. Genetics 208:439-445 (1987); Russell,P. W. & Orndorff, P. E., J. Bacteriol. 174:5923-5935 (1992)). Recently,it was shown that FimH might also form a thin tip-fibrillum at the endof the pili (Jones, C. H., et al., Proc. Nat. Acad. Sci. USA92:2081-2085 (1995)). The order of major and minor components in theindividual mature pili is very similar, indicating a highly orderedassembly process (Soto, G. E. & Hultgren, S. J., J. Bacteriol.181:1059-1071 (1999)).

P-pili of E. coli are of very similar architecture, have a diameter of6.8 nm, an axial hole of 1.5 nm and 3.28 subunits per turn (Bullitt &Makowski, Biophys. J. 74:623-632 (1998)). The 16.6 kDa PapA is the maincomponent of this pilus type and shows 36% sequence identity and 59%similarity to FimA (see Table 1). As in Type-1 pili the 36.0 kDa P-pilusadhesin PapG and specialized adapter proteins make up only a tinyfraction of total pilus protein. The most obvious difference to Type-1pili is the absence of the adhesin as an integral part of the pilus rod,and its exclusive localization in the tip fibrillium that is connectedto the pilus rod via specialized adapter proteins that Type-1 pili lack(Hultgren, S. J., et al., Cell 73:887-901 (1993)).

TABLE 1 Similarity and identity between several structural pilusproteins of Type-1 and P-pili (in percent). The adhesins were omitted.Similarity FimA PapA FimI FimF FimG PapE PapK PapH PapF Identity FimA 5957 56 44 50 44 46 46 PapA 36 49 48 41 45 49 49 47 FimI 35 31 56 46 40 4748 48 FimF 34 26 30 40 47 43 49 48 FimG 28 28 28 26 39 39 41 45 PapE 2523 18 28 22 43 47 54 PapK 24 29 25 28 22 18 49 53 PapH 22 26 22 22 23 2423 41 PapF 18 22 22 24 28 27 26 21

Type-1 pili are extraordinary stable hetero-oligomeric complexes.Neither SDS-treatment nor protease digestions, boiling or addition ofdenaturing agents can dissociate Type-1 pili into their individualprotein components. The combination of different methods like incubationat 100° C. at pH 1.8 was initially found to allow for thedepolymerization and separation of the components (Eshdat, Y., et al.,J. Bacteriol. 148:308-314 (1981); Brinton, C. C. J., Trans, N Y. Acad.Sci. 27:1003-1054 (1965); Hanson, A. S., et al., J. Bacteriol.,170:3350-3358 (1988); Klemm, P. & Krogfelt, K. A., “Type I fimbriae ofEscherichia coli,” in: Fimbriae. Klemm, P. (ed.), CRC Press Inc., (1994)pp. 9-26). Interestingly, Type-1 pili show a tendency to break atpositions where FimH is incorporated upon mechanical agitation,resulting in fragments that present a FimH adhesin at their tips. Thiswas interpreted as a mechanism of the bacterium to shorten pili to aneffective length under mechanical stress (Klemm, P. & Krogfelt, K. A.,“Type I fimbriae of Escherichia coli,” in: Fimbriae. Klemm, P. (ed.),CRC Press Inc., (1994) pp. 9-26). Despite their extraordinary stability,Type-1 pili have been shown to unravel partially in the presence of 50%glycerol; they lose their helical structure and form an extended andflexible, 2 nm wide protein chain (Abraham, S. N., et al., J. Bacteriol.174:5145-5148 (1992)).

P-pili and Type-1 pili are encoded by single gene clusters on the E.coli chromosome of approximately 10 kb (Klemm, P. & Krogfelt, K. A.,“Type I fimbriae of Escherichia coli,” in: Fimbriae. Klemm, P. (ed.),CRC Press Inc., (1994) pp. 9-26; Orndorff, P. E. & Falkow, S., J.Bacteriol. 160:61-66 (1984)). A total of nine genes are found in theType-1 pilus gene cluster, and 11 genes in the P-pilus cluster(Hultgren, S. J., et al., Adv. Prot. Chem. 44:99-123 (1993)). Bothclusters are organized quite similarly.

The first two fim-genes, fimB and fimE, code for recombinases involvedin the regulation of pilus expression (McClain, M. S., et al., J.Bacteriol. 173:5308-5314 (1991)). The main structural pilus protein isencoded by the next gene of the cluster, fimA (Klemm, P., Euro. J.Biochem. 143:395-400 (1984); Orndorff, P. E. & Falkow, S., J. Bacteriol.160:61-66 (1984); Orndorff, P. E. & Falkow, S., J. Bacteriol.162:454-457 (1985)). The exact role of fimI is unclear. It has beenreported to be incorporated in the pilus as well (Klemm, P. & Krogfelt,K. A., “Type I fimbriae of Escherichia coli,” in: Fimbriae. Klemm, P.(ed.), CRC Press Inc., (1994) pp. 9-26). The adjacent fimC codes not fora structural component of the mature pilus, but for a so-called piluschaperone that is essential for the pilus assembly (Klemm, P., Res.Microbiol. 143:831-838 (1992); Jones, C. H., et al., Proc. Nat. AcadSci. USA 90:8397-8401 (1993)).

The assembly platform in the outer bacterial membrane to which themature pilus is anchored is encoded by fimD (Klemm, P. & Christiansen,G., Mol. Gen, Genetics 220:334-338 (1990)). The three minor componentsof the Type-1 pili, FimF, FimG and FimH are encoded by the last threegenes of the cluster (Klemm, P. & Christiansen, G., Mol. Gen. Genetics208:439-445 (1987)). Apart from fimB and fimE, all genes encodeprecursor proteins for secretion into the periplasm via the sec-pathway.

The similarities between different pili following the chaperone/usherpathway are not restricted to their morphological properties. Theirgenes are also arranged in a very similar manner Generally the gene forthe main structural subunit is found directly downstream of theregulatory elements at the beginning of the gene cluster, followed by agene for an additional structural subunit (final in the case of Type-1pili and papH in the case of P-pili). PapH was shown and FimI issupposed to terminate pilus assembly (Hultgren, S. J., et al., Cell73:887-901 (1993)). The two proteins that guide the process of pilusformation, namely the specialized pilus chaperone and the outer membraneassembly platform, are located adjacently downstream. At the end of theclusters a variable number of minor pilus components including theadhesins are encoded. The similarities in morphological structure,sequence (see Table 1), genetic organization and regulation indicate aclose evolutionary relationship and a similar assembly process for thesecell organelles.

Bacteria producing Type-1 pili show a so-called phase-variation. Eitherthe bacteria are fully piliated or bald. This is achieved by aninversion of a 314 by genomic DNA fragment containing the fimA promoter,thereby inducing an “all on” or “all off” expression of the pilus genes(McClain, M. S., et al., J. Bacteriol. 173:5308-5314 (1991)). Thecoupling of the expression of the other structural pilus genes to fimAexpression is achieved by a still unknown mechanism. However, a widerange of studies elucidated the mechanism that influences the switchingbetween the two phenotypes.

The first two genes of the Type-1 pilus cluster, fimB and fimE encoderecombinases that recognize 9 by DNA segments of dyad symmetry thatflank the invertable fimA promoter. Whereas FimB switches pilation “on”,FimE turns the promoter in the “off” orientation. The up- ordown-regulation of either fimB or fimE expression therefore controls theposition of the so-called “fim-switch” (McClain, M. S., et al., J.Bacteriol. 173:5308-5314 (1991); Blomfield, I. C., et al., J. Bacteriol.173:5298-5307 (1991)).

The two regulatory proteins fimB and fimE are transcribed from distinctpromoters and their transcription was shown to be influenced by a widerange of different factors including the integration host factor (IHF)(Blomfield, I. C., et al., Mol. Microbiol. 23:705-717 (1997)) and theleucine-responsive regulatory protein (LRP) (Blomfield, I. C., et al.,J. Bacteriol. 175:27-36 (1993); Gally, D. L., et al., J. Bacteriol.175:6186-6193 (1993); Gally, D. L., et al., Microbiol. 21:725-738(1996); Roesch, R. L. & Blomfield, I. C., Mol. Microbiol, 27:751-761(1998)). Mutations in the former lock the bacteria either in “on” or“off” phase, whereas LRP mutants switch with a reduced frequency. Inaddition, an effect of leuX on pilus biogenesis has been shown. Thisgene is located in the vicinity of the fim-genes on the chromosome andcodes for the minor leucine tRNA species for the UUG codon. Whereas fimBcontains five UUG codons, fimE contains only two, and enhanced leuXtranscription might favor FimB over FimE expression (Burghoff, R. L., etal., Infect. Immun. 61:1293-1300 (1993); Newman, J. V., et al., FEMSMicrobiol. Lett. 122:281-287 (1994); Ritter, A., et al., Mol. Microbial,25:871-882 (1997)).

Furthermore, temperature, medium composition and other environmentalfactors were shown to influence the activity of FimB and FimE Finally, aspontaneous, statistical switching of the fimA promoter has beenreported. The frequency of this spontaneous switching is approximately10⁻³ per generation (Eisenstein, B. I., Science 214:337-339 (1981);Abraham, S. M., et al., Proc. Nat. Acad. Sci, USA 82:5724-5727 (1985)),but is strongly influenced by the above mentioned factors.

The genes fimI and fimC are also transcribed from the fimA promoter, butdirectly downstream of fimA a DNA segment with a strong tendency to formsecondary structure was identified which probably represents a partialtranscription terminator (Klemm, P., Euro. J. Biochem. 143:395-400(1984)); and is therefore supposed to severely reduce fimI and fimCtranscription. At the 3′ end of fimC an additional promoter controls thefimD transcription; at the 3′ end of fimD the last known fim promoter islocated that regulates the levels of FimF, FimG, and FimH. Thus, all ofthe minor Type-1 pili proteins are transcribed as a single mRNA (Klemm,P. & Krogfelt, K. A., “Type I fimbriae of Escherichia coli,” in:Fimbriae. Klemm, P. (ed.), CRC Press Inc., (1994) pp. 9-26). Thisensures a 1:1:1 stoichiometry on mRNA-level, which is probablymaintained on the protein level.

In the case of P-pili additional regulatory mechanisms were found whenthe half-life of mRNA was determined for different P-pilus genes. ThemRNA for papA was extraordinarily long-lived, whereas the mRNA for papB,a regulatory pilus protein, was encoded by short-lived mRNA(Naureckiene, S. & Uhlin. B. E., Mol. Microbiol. 21:55-68 (1996);Nilsson, P., et al., J. Bacterial. 178:683-690 (1996)).

In the case of Type-1 pili, the gene for the Type-1 pilus chaperone FimCstarts with a GTG instead of an ATG codon, leading to a reducedtranslation efficiency. Finally, analysis of the fimH gene revealed atendency of the fimH mRNA to form a stem-loop, which might severelyhamper translation. In summary, bacterial pilus biogenesis is regulatedby a wide range of different mechanisms acting on all levels of proteinbiosynthesis.

Periplasmic pilus proteins are generally synthesized as precursors,containing a N-terminal signal-sequence that allows translocation acrossthe inner membrane via the Sec-apparatus. After translocation theprecursors are normally cleaved by signal-peptidase I. Structural Type-1pilus subunits normally contain disulfide bonds, their formation iscatalyzed by DsbA and possibly DsbC and DsbG gene products.

The Type-1 pilus chaperone FimC lacks cysteine residues. In contrast,the chaperone of P-pili, PapD, is the only member of the pilus chaperonefamily that contains a disulfide bond, and the dependence of P-pili onDsbA has been shown explicitly (Jacob-Dubuisson, F., et al., Proc. Nat.Acad. Sci. USA 91:11552-11556 (1994)). PapD does not accumulate in theperiplasm of a

dsbA strain, indicating that the disturbance of the P-pilus assemblymachinery is caused by the absence of the chaperone (Jacob-Dubuisson,F., et al., Proc. Nat. Acad. Sci. USA 91:11552-11556 (1994)). This is inaccordance with the finding that Type-1 pili are still assembled in a

dsbA strain, albeit to reduced level (Hultgren, S. J., et al.,“Bacterial Adhesion and Their Assembly”, in: Escherichia coli andSalmonella, Neidhardt, F. C. (ed.) ASM Press, (1996) pp. 2730-2756).

Type-1 pili as well as P-pili are to 98% made of a single or mainstructural subunit termed FimA and PapA, respectively. Both proteinshave a size of ˜15.5 kDa. The additional minor components encoded in thepilus gene clusters are very similar (see Table 1). The similarities insequence and size of the subunits with the exception of the adhesinssuggest that all share an identical folding motif, and differ only withrespect to their affinity towards each other. Especially the N- andC-terminal regions of these proteins are well conserved and supposed toplay an important role in chaperone/subunit interactions as well as insubunit/subunit interactions within the pilus (Soto, G. E. & Hultgren,S. J., J. Bacteriol. 181:1059-1071 (1999)). Interestingly, the conservedN-terminal segment can be found in the middle of the pilus adhesins,indicating a two-domain organization of the adhesins where the proposedC-terminal domain, starting with the conserved motif, corresponds to astructural pilus subunit whereas the N-terminal domain was shown to beresponsible for recognition of host cell receptors (Hultgren, S. J., etal., Proc. Nat. Acad. Sci. USA 86:4357-4361 (1989); Haslam, D. B., etal., Mol. Microbiol. 14:399-409 (1994); Soto, G. E., et al., EMBO J.17:6155-6167 (1998)). The different subunits were also shown toinfluence the morphological properties of the pili. The removal ofseveral genes was reported to reduce the number of Type-1 or P-pili orto increase their length, (fimH, papG, papK, fimF, fimG) (Russell, P. W.& Orndorff, P. E., J. Bacteriol. 174:5923-5935 (1992); Jacob-Dubuisson,R., et al., EMBO J. 12:837-847 (1993); Soto, G. E. & Hultgren, S. J., J.Bacteriol. 181:1059-1071 (1999)); combination of the gene deletionsamplified these effects or led to a total loss of pilation(Jacob-Dubuisson, R., et al., EMBO J. 12:837-847 (1993)).

In non-fimbrial adhesive cell organelles also assembled viachaperones/usher systems such as Myf fimbriae and CS3 pili, theconserved C-terminal region is different. This indirectly proves theimportance of these C-terminal subunit segments for quaternaryinteractions (Hultgren, S. J., et al., “Bacterial Adhesion and TheirAssembly”, in: Escherichia coli and Salmonella, Neidhardt, F. C. (ed.)ASM Press, (1996) pp. 2730-2756).

Gene deletion studies proved that removal of the pilus chaperones leadsto a total loss of piliation in P-pili and Type-1 pili (Lindberg, F., etal., J. Bacteriol. 171:6052-6058 (1989); Klemm, P., Res. Microbiol.143:831-838 (1992); Jones, C. H., et al., Proc. Nat. Acad Sci. USA90:8397-8401 (1993)). Periplasmic extracts of a

fimC strain showed the accumulation of the main subunit FimA, but nopili could be detected (Klemm, P., Res. Microbiol. 143:831-838 (1992)).Attempts to over-express individual P-pilus subunits failed and onlyproteolytically degraded forms could be detected in the absence of PapD;in addition, the P-pilus adhesin was purified with the inner membranefraction in the absence of the chaperone (Lindberg, F., et al., J.Bacteriol. 171:6052-6058 (1989)). However, co-expression of thestructural pilus proteins and their chaperone allowed the detection ofchaperone/subunit complexes from the periplasm in the case of theFimC/FimH complex as well as in the case of different Pap-proteinsincluding the adhesin PapG and the main subunit PapA (Tewari, R., etal., J. Biol. Chem. 268:3009-3015 (1993); Lindberg, F., et al., J.Bacteriol. 171:6052-6058 (1989)). The affinity of chaperone/subunitcomplexes towards their assembly platform has also been investigated invitro and was found to differ strongly (Dodson et al., Proc. Natl. Acad.Sci. USA 90:3670-3674 (1993)). From these results the followingfunctions were suggested for the pilus chaperones.

They are assumed to recognize unfolded pilus subunits, prevent theiraggregation and to provide a “folding template” that guides theformation of a native structure.

The folded subunits, which after folding display surfaces that allowsubunit/subunit interactions, are then expected to be shielded frominteracting with other subunits, and to be kept in a monomeric,assembly-competent state.

Finally, the pilus chaperones are supposed to allow a triggered releaseof the subunits at the outer membrane assembly location, and, by doingso with different efficiency, influence the composition and order of themature pili (see also the separate section below).

After subunit release at the outer membrane, the chaperone is free foranother round of substrate binding, folding assistance, subunittransport through the periplasm and specific delivery to the assemblysite. Since the periplasm lacks energy sources, like ATP, the wholepilus assembly process must be thermodynamically driven(Jacob-Dubuisson, F., et al., Proc. Nat. Acad. Sci. USA 91:11552-11556(1994)). The wide range of different functions attributed to the piluschaperones would implicate an extremely fine tuned cascade of steps.

Several findings, however, are not readily explained with the model ofpilus chaperone function outlined above. One example is the existence ofmultimeric chaperone/subunit complexes (Striker, R. T., et al., J. Biol.Chem. 269:12233-12239 (1994)), where one chaperone binds subunit dimersor trimers. It is difficult to imagine a folding template that can be“double-booked”. The studies on the molecular details ofchaperone/subunit interaction (see below) partially supported thefunctions summarized above, but also raised new questions.

All 31 periplasmic chaperones identified by genetic studies or sequenceanalysis so far are proteins of approximately 25 kDa with conspicuouslyhigh pI values around 10. Ten of these chaperones assist the assembly ofrod-like pili, four are involved in the formation of thin pili, ten areimportant for the biogenesis of atypically thin structures (includingcapsule-like structures) and two adhesive structures have not beendetermined so far (Holmgren, A., et al., EMBO J. 11:1617-1622 (1992);Bonci, A., et al., J. Mol. Evolution 44:299-309 (1997); Smyth, C. J., etal., FEMS Immun. Med Microbiol. 16:127-139 (1996); Hung, D. L. &Hultgren, S. J., J. Struct, Biol. 124:201-220 (1998)). The pairwisesequence identity between these chaperones and PapD ranges from 25 to56%, indicating an identical overall fold (Hung, D. L., et al., EMBO J.15:3792-3805 (1996)).

The first studies on the mechanism of chaperone/substrate recognitionwas based on the observation that the C-termini of all known piluschaperones are extremely similar. Synthetic peptides corresponding tothe C-termini of the P-pilus proteins were shown to bind to PapD inELISA assays (Kuehn, M. J., et al., Science 262:1234-1241 (1993)). Mostimportantly, the X-ray structures of two complexes were solved in whichPapD was co-crystallized with 19-residue peptides corresponding to theC-termini of either the adhesin PapG or the minor pilus component PapK(Kuehn, M. J., et al., Science 262:1234-1241 (1993); Soto, G. E., etal., EMBO J. 17:6155-6167 (1998)). Both peptides bound in an extendedconformation to a β-strand in the N-terminal chaperone domain that isoriented towards the inter-domain cleft, thereby extending a β-sheet byan additional strand. The C-terminal carboxylate groups of the peptideswere anchored via hydrogen-bonds to Arg8 and Lys112, these two residuesare invariant in the family of pilus chaperones. Mutagenesis studiesconfirmed their importance since their exchange against alanine resultedin accumulation of non-functional pilus chaperone in the periplasm(Slonim, L. N., et al., EMBO J. 11:4747-4756 (1992)). The crystalstructure of PapD indicates that neither Arg8 nor Lys112 is involved instabilization of the chaperone, but completely solvent exposed(Holmgren, A. & Branden, C. I., Nature 342:248-251 (1989)). On thesubstrate side the exchange of C-terminal PapA residues was reported toabolish P-pilus formation, and similar experiments on the conservedC-terminal segment of the P-pilus adhesin PapG prevented itsincorporation into the P-pilus (Hultgren, S. J., et al., “BacterialAdhesion and Their Assembly”, in: Escherichia coli and Salmonella,Neidhardt, F. C. (ed.) ASM Press, (1996) pp. 2730-2756). All evidencetherefore indicated pilus subunit recognition via the C-terminalsegments of the subunits.

A more recent study on C-terminal amino acid exchanges of the P-pilusadhesin PapG gave a more detailed picture. A range of amino acidsubstitutions at the positions −2, −4, −6, and −8 relative to theC-terminus were tolerated, but changed pilus stability (Soto, G. E., etal., EMBO J. 17:6155-6167 (1998)).

Still, certain problems arise when this model is examined more closely.Adhesive bacterial structures not assembled to rigid, rod-like pili lackthe conserved C-terminal segments (Hultgren, S. J., et al., “BacterialAdhesion and Their Assembly”, in: Escherichia coli and Salmonella,Neidhardt, F. C. (ed.) ASM Press, (1996) pp. 2730-2756), even thoughthey are also dependent on the presence of related pilus chaperones.This indicates a different general role for the C-terminal segments ofpilus subunits, namely the mediation of quaternary interactions in themature pilus. Moreover, the attempt to solve the structure of aC-terminal peptide in complex with the chaperone by NMR was severelyhampered by the weak binding of the peptide to the chaperone (Walse, B.,et al., FEBS Lett. 412:115-120 (1997)); whereas an essentialcontribution of the C-terminal segments for chaperone recognitionimplies relatively high affinity interactions.

An additional problem arises if the variability between the differentsubunits are taken into account. Even though the C-terminal segments areconserved, a wide range of conservative substitutions is found. Forexample, 15 out of 19 amino acid residues differ between the twopeptides co-crystallized with PapD (Soto, G. E., et al., EMBO J.17:6155-6167 (1998)). This has been explained by the kind of interactionbetween chaperone and substrate, that occurs mainly via backboneinteractions and not specifically via side-chain interactions. Thenagain, the specificity of the chaperone for certain substrates is notreadily explained. On the contrary to the former argument, the conservedresidues have been taken as a proof for the specificity (Hultgren, S.J., et al., “Bacterial Adhesion and Their Assembly”, in: Escherichiacoli and Salmonella, Neidhardt, F. C. (ed.) ASM Press, (1996) pp.2730-2756).

The outer membrane assembly platform, also termed “usher” in theliterature, is formed by homo-oligomers of FimD or PapC, in the case ofType-1 and P-pili, respectively (Klemm, P. & Christiansen, G., Mol. Gen,Genetics 220:334-338 (1990); Thanassi, D. G., et al., Proc. Nat. Acad.Sci. USA 95:3146-3151 (1998)). Studies on the elongation of Type-1fimbriae by electron microscopy demonstrated an elongation of the pilusfrom the base (Lowe, M. A., et al., J. Bacteriol. 169:157-163 (1987)).In contrast to the secretion of unfolded subunits into the periplasmicspace, the fully folded proteins have to be translocated through theouter membrane, possibly in an oligomeric form (Thanassi, D. G., et al.,Proc. Nat. Acad. Sci. USA 9 5:3146-3151 (1998)). This requires first amembrane pore wide enough to allow the passage and second a transportmechanism that is thermodynamically driven (Jacob-Dubuisson, F., et al.,J. Biol. Chem. 269:12447-12455 (1994)).

FimD expression alone was shown to have a deleterious effect onbacterial growth, the co-expression of pilus subunits could restorenormal growth behavior (Klemm, P. & Christiansen, G., Mol. Gen, Genetics220:334-338 (1990)). Based on this it can be concluded that the ushersprobably form pores that are completely filled by the pilus. Electronmicroscopy on membrane vesicles in which PapC had been incorporatedconfirmed a pore-forming structure with an inner diameter of 2 nm(Thanassi, D. G., et al., Proc. Nat. Acad. Sci. USA 95:3146-3151(1998)). Since the inner diameter of the pore is too small to allow thepassage of a pilus rod, it has been suggested that the helicalarrangement of the mature pilus is formed at the outside of thebacterial surface. The finding that glycerol leads to unraveling of piliwhich then form a protein chain of approximately 2 nm is in goodagreement with this hypothesis, since an extended chain of subunitsmight be formed in the pore as a first step (Abraham, S. N., et al., J.Bacteriol. 174:5145-5148 (1992); Thanassi, D. G., et al., Proc. Nat.Acad. Sci. USA 95:3146-3151 (1998)). The formation of the helical pilusrod at the outside of the bacterial membrane might then be the drivingforce responsible for translocation of the growing pilus through themembrane.

It has also been demonstrated that the usher proteins of Type-1 andP-pili form ternary complexes with chaperone/subunit complexes withdifferent affinities (Dodson, K. W., et al., Proc. Nat. Acad. Sci. USA90:3670-3674 (1993); Saulino, E. T., et al., EMBO J. 17:2177-2185(1998)). This was interpreted as “kinetic partitioning” that allows adefined order of pilus proteins in the pilus. Moreover, it has beensuggested that structural proteins might present a binding surface onlycompatible with one other type of pilus protein; this would be anothermechanism to achieve a highly defined order of subunits in the maturepilus (Saulino, E. T., et al., EMBO J. 17:2177-2185 (1998)).

B. Production of Type-1 Pili from Escherichia coli

E. coli strain W3110 was spread on LB (10 g/L tryptone, 5 g/L yeastextract, 5 g/L NaCl, pH 7.5, 1% agar (w/v)) plates and incubated at 37°C. overnight. A single colony was then used to inoculate 5 ml of LBstarter culture (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH7.5). After incubation for 24 hours under conditions that favor bacteriathat produce Type-1 pili (37° C., without agitation) 5 shaker flaskscontaining 1 liter LB were inoculated with one milliliter of the starterculture. The bacterial cultures were then incubated for additional 48 to72 hours at 37° C. without agitation. Bacteria were then harvested bycentrifugation (5000 rpm, 4° C., 10 minutes) and the resulting pelletwas resuspended in 250 milliliters of 10 mM Tris/HCl, pH 7.5. Pili weredetached from the bacteria by 5 minutes agitation in a conventionalmixer at 17.000 rpm. After centrifugation for 10 minutes at 10,000 rpmat 4° C. the pili containing supernatant was collected and 1 M MgCl₂ wasadded to a final concentration of 100 mM. The solution was kept at 4° C.for 1 hour, and the precipitated pili were then pelleted bycentrifugation (10,000 rpm, 20 minutes, 4° C.). The pellet was thenresuspended in 10 mM HEPES, pH 7.5, and the pilus solution was thenclarified by a final centrifugation step to remove residual cell debris.

C. Coupling of FLAG to Purified Type-1 Pili of E. coli Usingm-Maleimidonbenzoyl-N-Hydroxysulfosuccinimide Ester (Sulfo-MBS)

600 μl of a 95% pure solution of bacterial Type-1 pili (2 mg/ml) wereincubated for 30 minutes at room temperature with the heterobifunctionalcross-linker sulfo-MBS (0.5 mM). Thereafter, the mixture was dialyzedovernight against 1 liter of 50 mM Phosphate buffer (pH 7.2) with 150 mMNaCl to remove free sulfo-MBS. Then 500 μl of the derivatized pili (2mg/ml) were mixed with 0.5 mM FLAG peptide (containing an amino-terminalCysteine) in the presence of 10 mM EDTA to prevent metal-catalyzedsufhydryloxidation. The non-coupled peptide was removed bysize-exclusion-chromatography.

Example 34 Construction of an Expression Plasmid for the Expression ofType-1 Pili of Escherichia coli

The DNA sequence disclosed in GenBank Accession No. U14003, the entiredisclosure of which is incorporated herein by reference, contains all ofthe Escherichia coli genes necessary for the production of type-1 pilifrom nucleotide number 233947 to nucleotide number 240543 (the fim genecluster). This part of the sequences contains the sequences for thegenes fimA, fimI, fimC, fimD, fimF, fimG, and fimH. Three different PCRswere employed for the amplification of this part of the E. coli genomeand subsequent cloning into pUC19 (GenBank Accession Nos. L09137 andX02514) as described below.

The PCR template was prepared by mixing 10 ml of a glycerol stock of theE. coli strain W3110 with 90 ml of water and boiling of the mixture for10 minutes at 95° C., subsequent centrifugation for 10 minutes at 14,000rpm in a bench top centrifuge and collection of the supernatant.

Ten ml of the supernatant were then mixed with 50 pmol of a PCR primerone and 50 pmol of a PCR primer two as defined below. Then 5 ml of a10×PCR buffer, 0.5 ml of Taq-DNA-Polymerase and water up to a total of50 ml were added. All PCRs were carried out according to the followingscheme: 94° C. for 2 minutes, then 30 cycles of 20 seconds at 94° C., 30seconds at 55° C., and 2 minutes at 72° C. The PCR products were thenpurified by 1% agarose gel-electrophoresis.

Oligonucleotides with the following sequences with were used to amplifythe sequence from nucleotide number 233947 to nucleotide number 235863,comprising the fimA, fimI, and fimC genes:

(SEQ ID NO: 196) TAGATGATTACGCCAAGCTTATAATAGAAATAGTTTTTTGAAAGGAAAGCAGCATG and (SEQ ID NO: 197)GTCAAAGGCCTTGTCGACGTTATTCCATTACGCCCGTCATTTTG G

These two oligonucleotides also contained flanking sequences thatallowed for cloning of the amplification product into puc19 via therestriction sites HindIII and SalI. The resulting plasmid was termedpFIMAIC (SEQ ID NO:198).

Oligonucleotides with the following sequences with were used to amplifythe sequence from nucleotide number 235654 to nucleotide number 238666,comprising the fimD gene:

(SEQ ID NO: 199) AAGATCTTAAGCTAAGCTTGAATTCTCTGACGCTGATTAACC and(SEQ ID NO: 200) ACGTAAAGCATTTCTAGACCGCGGATAGTAATCGTGCTATC.

These two oligonucleotides also contained flanking sequences thatallowed for cloning of the amplification product into puc19 via therestriction sites HindIII and XbaI, the resulting plasmid was termedpFIMD (SEQ ID NO:201).

Oligonucleotides with the following sequences with were used to amplifythe sequence from nucleotide number 238575 nucleotide number 240543,comprising the fimF, fimG, and fimH gene:

(SEQ ID NO: 202) AATTACGTGAGCAAGCTTATGAGAAACAAACCTTTTTATC and(SEQ ID NO: 203) GACTAAGGCCTTTCTAGATTATTGATAAACAAAAGTCACGC.

These two oligonucleotides also contained flanking sequences thatallowed for cloning of the amplification product into puc19 via therestriction sites HindIII and XbaI; the resulting plasmid was termedpFIMFGH. (SEQ ID NO:204).

The following cloning procedures were subsequently carried out togenerate a plasmid containing all the above-mentioned fim-genes:

pFIMAIC was digested EcoRI and HindIII (2237-3982), pFIMD was digestedEcoRI and SstII (2267-5276), pFIMFGH was digested SstII and HindIII(2327-2231). The fragments were then ligated and the resulting plasmid,containing all the fim-genes necessary for pilus formation, was termedpFIMAICDFGH (SEQ ID NO:205).

Example 35 Construction of an Expression Plasmid for Escherichia coliType-1 Pili that Lacks the Adhesion FimH

The plasmid pFIMAICDFGH (SEQ ID NO:205) was digested with Kpn1, afterwhich a fragment consisting of nucleotide numbers 8895-8509 was isolatedby 0.7% agarose gelelectrophoresis and circularized by self-ligation.The resulting plasmid was termed pFIMAICDFG (SEQ ID NO: 206), lacks thefimH gene and can be used for the production of FIMH-free type-1 pili.

Example 36 Expression of Type-1 Pili Using the Plasmid pFIMAICDFGH

E. coli strain W3110 was transformed with pFIMAICDFGH (SEQ ID NO:205)and spread on LB (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH7.5, 1% agar (w/v)) plates containing 100 μg/ml ampicillin and incubatedat 37° C. overnight. A single colony was then used to inoculate 50 ml ofLB-glucose starter culture (10 g/L tryptone, 5 g/L yeast extract, 1%(w/v) glucose, 5 g/L NaCl, pH 7.5, 100 mg/ml ampicillin). Afterincubation for 12-16 hours at 37° C. at 150 rpm, a 5 liter shaker flaskscontaining 2 liter LB-glucose was inoculated with 20 milliliter of thestarter culture. The bacterial cultures were then incubated foradditional 24 at 37° C. with agitation (150 rpm). Bacteria were thenharvested by centrifugation (5000 rpm, 4° C., 10 minutes) and theresulting pellet was resuspended in 250 milliliters of 10 mM Tris/HCl,pH 8. Pili were detached from the bacteria by agitation in aconventional mixer at 17,000 rpm for 5 minutes. After centrifugation for10 minutes at 10,000 rpm, 1 hour, ° C. the supernatant containing piliwas collected and 1 M MgCl₂ was added to a final concentration of 100mM. The solution was kept at 4° C. for 1 hour, and precipitated piliwere then pelleted by centrifugation (10,000 rpm, 20 minutes, 4° C.).The pellet was then resuspended in 10 mM HEPES, 30 mM EDTA, pH 7.5, for30 minutes at room temperature, and the pilus solution was thenclarified by a final centrifugation step to remove residual cell debris.The preparation was then dialyzed against 20 mM HEPES, pH 7.4.

Example 37 Coupling of IgE Epitopes and Mimotopes to Type-1 Pili ofEscherichia coli

A 66 μl aliquot of a 100 uM solution of the heterobifunctionalcross-linker sulfa-MBS was added to 400 μl of a 95% pure solution ofbacterial Type-1 pili (2.5 mg/ml, 20 mM HEPES, pH 7.4) and subsequentlyincubated for 45 minutes at room temperature with agitation. Thereafter,the excess of sulfa-MBS was removed by size exclusion chromatographyusing a PD-10 column. Alternatively, the cross-linker can be removed bydialysis. Then either 1.3 μl of a solution containing 1.1 mg/ml peptideCe3epi (CGGVNLTWSRA SG (SEQ ID NO:207)), or peptide Ce3Mim(CGGVNLPWSFGLE (SEQ ID NO:208) was added to 1 ml aliquots of thederivatized pili (1-1.25 mg/ml, 20 mM HEPES pH 7.4). The samples wereincubated at room temperature for 4 h and non-coupled peptide wasremoved by dialysis against 2 times 2 l of a buffer containing 20 mMHEPES (pH 7.4). Alternatively, the non-coupled peptide can be removed bysize-exclusion chromatography.

Example 38 Immunization of Mice with a Bee Venom Phospholipase A₂ (PLA₂)Fusion Protein Coupled to Qβ Capsid Protein

A. Preparation of an Alternative Vector for Cytoplasmic Expression ofthe Catalytically Inactive Variant of the PLA₂ Gene Fused to the AminoAcid Sequence AAASGGCGG (SEQ ID NO: 209)

The PLA₂ gene construct of example 9 was amplified by PCR frompAV3PLAfos using oligos ecori_Nde1_p1a (sequence below) and PLA-Cys-hind(Example 29). For the reaction, 100 pmol of each oligo, and about 1 μgof PAV3PLAfos DNA were used in the 50 μl reaction mixtures with 1.2units of Pfx DNA polymerase (Gibco), 1 mM MgSO₄, 200 μM dNTPS and Pfxenhancer solution (Gibco) diluted ten times. For the reaction,temperature cycling was carried out as follows: 94° C. for 2 minutes, 5cycles of 92° C. (0.5 minutes), 58° C. (0.5 minutes), 68° C. (1 minute);25 cycles of 92° C. (0.5 minutes), 63° C. (0.5 minutes), 68° C. (1minute). The PCR product was purified by agarose gel electrophoresis andsubsequent isolation of the fragment using the Qiagen Qiaquick Kit,digested with enzymes Nde1 and HindIII, and cloned into the PET11avector (Novagen) digested with the same enzymes.

Oligos:

ecorl_Ndel_pla: (SEQ ID NO: 214)TAACCGAATTCAGGAGGTAAAAACATATGGC TATCATCTACC.

The vector encoded a fusion protein having the amino acid sequence

(SEQ ID NO: 210) MAIIYPGTLWCGHGNKSSGPNELGRFKHTDACCRTQDMCPDVMSAGESKHGLTNTASHTRLSCDCDDKFYDCLKNSADTISSYFVGKMYFNLIDTKCYKLEHPVTGCGERTEGRCLHYTVDKSKPKVYQWFDLRKYAAASGGCGG.

Coupling of PLA₂Fusion Protein to Qβ Capsid Protein

A solution of 600 μl of Qβ capsid protein (2 mg/ml in 20 mM Hepes, pH7.4) was reacted with 176 μl Sulfo-MBS (13 mg/ml in H₂O) for 60 minutesat room temperature, and dialyzed against 1 L of 20 mM Hepes pH 7.4 0/Nat 4° C. The next day, 500 μl of a PLA2 solution (2.5 mg/ml) containing0.1 mM DTT were desalted over a 5 ml Hi-Trap column (Pharmacia). Reducedand desalted PLA₂ (60 μl, of a solution of approx. 0.5 mg/ml) was mixedwith activated and dialyzed Qβ capsid (25 μl of a 1.5 mg/ml solution)and reacted for four hours at room temperature.

1 Capsids of 25-30 nm diameter are clearly visible in electronmicroscopy images of Qβ capsid protein taken both before and aftercoupling to PLA₂.

C. Immunization of Mice with PLA₂ Coupled to Qβ Capsid Protein

Female Balb/c mice were immunized intravenously on day 0 with 50 μg Qβcapsid coupled to PLA₂, and boosted on day 14 with the same amount ofantigen. Mice were bled on day 20 and sera analyzed in an ELISA. A titerof 1:5000 against PLA₂ was obtained.

Example 39 Coupling of IgE Mimotopes and Epitopes to Qβ Capsid Protein

Human IgE epitopes having the following amino acid sequences werecoupled to Qβ capsid protein using the N-terminal cysteine residue:

(SEQ ID NO: 207) Ce3epitope: CGGVNLTWSRASG (SEQ ID NO: 208)Ce3mimotope: CGGVNLPWSFGLE

The coupling reaction was performed using Qβ capsid protein activatedwith Sulfo-MBS and subsequently dialyzed to remove excess crosslinker.The respective epitope or mimotope was diluted into the reaction mixturecontaining the activated Qβ capsid, and left to react for 4 hours atroom temperature. The reaction mixture was finally dialyzed for 4 hoursagainst PBS, and injected into mice.

The following circular mimotope was also coupled to Qβ capsid protein:Ce4mimotope: GEFCINHRGYWVCGDPA (SEQ ID NO:211).

The mimotope was first reacted with the chemical groupN-succinimidyl-S-acetylthioacetate (SATA), in order to introduce aprotected sulfhydryl group into the mimotope. The protecting group wassubsequently removed by treatment with hydroxylamine, and immediatelyreacted with activated Qβ capsid protein, for 4 hours at roomtemperature. The reaction mixture was finally dialyzed for 4 hours, andinjected into mice.

Example 40 Immunization of Mice with HBcAg-Lys Coupled to M2 Peptide

A. Coupling of M2 Peptide to HBcAg-Lys Capsid Protein

Synthetic M2 peptide, corresponding to an N-terminal fragment of theInfluenza M2 protein with a cysteine residue at its C-terminus(SLLTEVETPIRNEWGCRCNGSSDGGGC (SEQ ID NO:212)) was chemically coupled topurified HBcAg-Lys particles in order to elicit an immune responseagainst the M2 peptide. Sulfo-MBS (232 μl, 3 mM) was reacted with asolution of 1.4 ml HBcAg-Lys (1.6 mg/ml) in PBS. The mixture wasdialyzed overnight against phosphate buffered saline (PBS). M2 peptidewas diluted to a concentration of 24 mg/ml in DMSO; 5 μl of thissolution was diluted in 300 μl PBS, 188 μl of which was added to 312 μlof the dialyzed activated HBcAg-Lys solution. EDTA (10 μl of a 1 Msolution) was also added to the reaction mixture, after which thereaction was allowed to proceed for 4 hours at room temperature.

Immunization of Mice with HBcAg-Lys Coupled to M2 Peptide

Female Balb/c mice were immunized intravenously on day 0 with 50 ngHBcAg-Lys-M2 or M2 peptide alone and boosted 10 days later with the sameamount of antigen. After another 10 days, the mice were infectedintranasally with Influenza virus (50 pfu, PR/8) and survival ofinfected mice was monitored. In addition, viral titers were determinedin the lung. Mice primed with M2-HBcAg-Lys were fully protected and hadeliminated the virus by day 7.

Example 41 Coupling of M2 Peptide to Pili, Qβ and Cys-Free HbcAg-CapsidProtein and Comparison of the Antibody Titer Obtained by Immunization ofMice with these Coupled Pili and Capsids with the Titer Obtained byImmunizing Mice with an N-Terminal Fusion Protein of the M2 Peptide toHbcAg1-183

A. Coupling of M2 Peptide to Pili, Q13- and Cys-Free HbcAg-CapsidProtein

Qβ:

A solution of 1 ml of 1 mg/ml Qβ capsid protein in 20 mM Hepes. 150 mMNaCl pH 7.2 was reacted for 30 minutes with 93 μl of a solution of 13mg/ml Sulfo-MBS (Pierce) in H₂O at RT on a rocking shaker. The reactionsolution was subsequently dialyzed overnight against 2 L of 20 mM hepes,150 mM NaCl, pH 7.2. The dialyzed reaction mixture was then reacted with58.8 μl of a 25 mM stock solution of M2 peptide (SEQ ID NO:212) in DMSOfor four hours at RT on a rocking shaker. The reaction mixture wassubsequently dialyzed against 2 liters of 20 mM Hepes, 150 mM NaCl, pH7.2 overnight at 4° C.

Cys-Free HbcAg:

A solution of 1.25 ml of 0.8 mg/ml cys-free HbcAg capsid protein(example 31) in PBS, pH 7.2 was reacted for 30 minutes with 93 μl of asolution of 13 mg/ml Sulfo-MBS (Pierce) in H₂O at RT on a rockingshaker. The reaction solution was subsequently dialyzed overnightagainst 2 L of 20 mM Hepes, 150 mM NaCl, pH 7.2. The dialyzed reactionmixture was then reacted with 58.8 μl of a 25 mM stock solution of M2peptide (SEQ ID NO:212) in DMSO for four hours at RT on a rockingshaker. The reaction mixture was subsequently dialyzed against 2 litersof 20 mM hepes, 150 mM NaCl, ph 7.2 overnight at 4° C.

Pili:

A solution of 400 μl of 2.5 mg/ml pili protein in 20 mM Hepes, pH 7.4,was reacted for 45 minutes with 60 μl of a 100 mM Sulfo-MBS (Pierce)solution in (H₂O) at RT on a rocking shaker. The reaction mixture wasdesalted on a PD-10 column (Amersham-Pharmacia Biotech), and the secondfraction of 500 μl protein elating from the column (containingapproximately 1 g protein) was reacted with 58.8 μl of a 25 mM stocksolution of M2 peptide (SEQ ID NO:212) in DMSO for four hours at RT on arocking shaker. The reaction mixture was subsequently dialyzed against 2liters of 20 mM Hepes, 150 mM NaCl, pH 7.2 overnight at 4° C.

Genetic Fusion of the M2 Peptide to HbcAg1-183

M2 genetically fused to Hbc: M2 was cloned at the N-terminus of Hbc aspublished by Neirynck et. al. Nature Medicine 5: 1157 (1999). MD-HBc wasexpressed in E. coli and purified by gel chromatography. The presence ofthe M2 peptide at the N-terminus of M2-HBc was confirmed by Edmansequencing.

Immunization of Mice:

Female Balb/c mice were vaccinated with M2 peptide coupled to pili, Qβand cys-free HbcAg protein and with M2 peptide genetically fused to Hbcimmunogen without the addition of adjuvants. 35 μg protein of eachsample were injected intraperitoneally on day 0 and day 14. Mice werebled on day 27 and their serum analyzed using a M2-peptide specificELISA.

ELISA

10 μg/ml M2 peptide coupled to RNAse was coated on an ELISA plate. Theplate was blocked then incubated with serially diluted mouse sera. Boundantibodies were detected with enzymatically labeled anti-mouse IgGantibody. As a control, preimmune sera were also tested. Control ELISAexperiments using sera from mice immunized with unrelated peptidescrosslinked to Hbc or other carriers showed the antibodies detected werespecific for the M2 peptide. The results are shown in FIGS. 27 A and B.

Example 42 Coupling of Angiotensin I and Angiotensin II Peptides to Qβand Immunization of Mice with Qβ-Angiotensin Peptide Vaccines

A. Coupling of Angiotensin I and Angiotensin II Peptides to Qβ CapsidProtein

The following angiotensin peptides were chemically synthesized:CGGDRVYIHPF (“Angio I”), CGGDRVYIHPFHL (“Angio II”), DRVYIHPFHLGGC(“Angio III”), CDRVYIHPFHL (“Angio IV”) and used for chemical couplingto Qβ as described in the following.

A solution of 5 ml of 2 mg/ml Qβ capsid protein in 20 mM Hepes. 150 mMNaCl pH 7.4 was reacted for 30 minutes with 507 μl of a solution of 13mg/ml Sulfo-MBS (Pierce) in H₂O at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. 665 ml of the dialyzedreaction mixture was then reacted with 2.8 ml of each of thecorresponding 100 mM peptide stock solution (in DMSO) for two hours at25° C. on a rocking shaker. The reaction mixture was subsequentlydialyzed 2×2 hours against 2 liters of 20 mM Hepes, 150 mM NaCl, pH 7.4at 4° C.

Immunization of Mice:

Female Balb/c mice were vaccinated with one of the four angiotensinpeptides coupled to Qβ capsid protein without the addition of adjuvants.50 μg of total protein of each sample was diluted in PBS to 200 ml andinjected subcutaneously (100 ml on two ventral sides) on day 0 and day14. Mice were bled retroorbitally on day 21 and their serum was analyzedusing a angiotensin-specific ELISA.

ELISA

All four angiotensin peptides were individually coupled to bovine RNAseA using the chemical cross-linker sulfo-SPDP. ELISA plates were coatedwith coupled RNAse preparations at a concentration of 10 mg/ml. Theplates were blocked and then incubated with serially diluted mouse sera.Bound antibodies were detected with enzymatically labeled anti-mouse IgGantibody. As a control, preimmune sera of the same mice were alsotested. Control ELISA experiments using sera from mice immunized withunrelated peptides crosslinked to Qβ or other carriers showed that theantibodies detected were specific for the respective peptide. Theresults are shown in FIG. 8A-8D.

FIGS. 8A, 8B, 8C and 8D, respectively, show ELISA analyses of IgGantibodies specific for “Angio I”, “Angio II”, “Angio III”, and “AngioIV”, respectively, in sera of mice immunized against Angio I-IV coupledto Qβ capsid protein. Qβ-Angio I, Qβ-Angio II, Qβ-Angio III and Qβ-AngioIV, as used in the figures, stand for the vaccine injected in the mice,from which the sera are derived in accordance with above definition ofthe angiotensin peptides.

Female Balb/c mice were vaccinated subcutaneously with 50 mg of vaccinein PBS on day 0 and day 14. IgG antibodies in sera of mice vaccinatedwith Qβ-Angio I, Qβ-Angio II, Qβ-Angio III and Qβ-Angio IV were measuredon day 21 against all four peptides (coupled to RNAse A), i.e. against“Angio I” (FIG. 8A), “Angio II” (FIG. 8B), “Angio III” (FIG. 8C), and“Angio IV” (FIG. 8D). As a control, pre-immune sera from the same micewere analyzed. Results for indicated serum dilutions are shown asoptical density at 450 nm. The average of three mice each (includingstandard deviations) is shown. All vaccinated mice made high IgGantibody titers against all four peptides tested. Noangiotensin-specific antibodies were detected in the controls(pre-immune mice).

Example 43 Coupling of Angiotensin I and Angiotensin II Peptides toHBcAg-149-Lys-2Cys-Mut, i.e. Cys-Free HBcAg

The following angiotensin peptides were chemically synthesized:CGGDRVYIHPF (“Angio I”), CGGDRVYIHPFHL (“Angio II”), DRVYIHPFHLGGC(“Angio III”), CDRVYIHPFHL (“Angio IV”) and are used for chemicalcoupling to HBcAg-149-lys-2cys-Mut, i.e. cys-free HBcAg.

A solution of 1.25 ml of 0.8 mg/ml HBcAg-149-lys-2cys-Mut capsid protein(cf. Example 31) in PBS, pH 7.4 is reacted for 30 minutes with 93 μl ofa solution of 13 mg/ml Sulfo-MBS (Pierce) in H₂O at 25° C. on a rockingshaker. The reaction solution is subsequently dialyzed overnight against2 L of 20 mM Hepes, 150 mM NaCl, pH 7.4. After buffer exchange thereaction solution is dialyzed for another 2 hours. The dialyzed reactionmixture is then reacted with 1.8 μl of a 100 mM peptide stock solution(in DMSO) for 2 hours at 25° C. on a rocking shaker. The reactionmixture is subsequently dialyzed against 2 liters of 20 mM Hepes, 150 mMNaCl, ph 7.4 overnight at 4° C. followed by buffer exchange and another2 hours of dialysis.

Example 44 Coupling of Angiotensin I and Angiotensin II Peptides toType-1 Pili of E. coli

The following angiotensin peptides were chemically synthesized:CGGDRVYIHPF (“Angio I”), CGGDRVYIHPFHL (“Angio II”), DRVYIHPFHLGGC(“Angio III”), CDRVYIHPFHL (“Angio IV”) and are used for chemicalcoupling to Type-1 pili of E. coli.

A solution of 400 μl of 2.5 mg/ml Type-1 pili of E. coli in 20 mM Hepes,pH 7.4, is reacted for 60 minutes with 60 μl of a 100 mM Sulfo-MBS(Pierce) solution in (H₂O) at RT on a rocking shaker. The reactionmixture is desalted on a PD-10 column (Amersham-Pharmacia Biotech), Theprotein-containing fractions eluating from the column are pooled(containing approximately 1 mg protein, i.e. derivatized pili) andreacted with a three-fold molar excess of peptide. For example, to 500ul eluate containing approximately 1 mg derivatized pili, 2.34 ul of a100 mM peptide stock solution (in DMSO) is added. The mixture isincubated for four hours at 25° C. on a rocking shaker and subsequentlydialyzed against 2 liters of 20 mM Hepes, 150 mM NaCl, pH 7.2 overnightat 4° C.

Example 45 Coupling of Der p I Peptides to Qβ and Immunization of Micewith Qβ-Der p I Vaccines

Coupling of Der p I Peptides to Qβ Capsid Protein

The following peptides derived from the house dust mite allergen Der p Iwere chemically synthesized: CGNQSLDLAEQELVDCASQHGCH (“Der p I p52”; aa52-72, with an additional cysteine-glycine linker at the N terminus),CQIYPPNANKIREALAQTHSA (“Der p 1 p117”; aa 117-137). These peptides wereused for chemical coupling to Qβ as described below.

1 ml of a solution consisting of 2 mg/ml Qβ capsid protein in 20 mMHepes, 150 mM NaCl, pH 7.4 was reacted for 30 minutes with 102 μl of asolution of 13 mg/ml Sulfo-MBS (Pierce) in H₂O at 25° C. on a rockingshaker. The reaction solution was subsequently dialyzed twice for 2hours against 2 L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. 440 μl ofthe dialyzed reaction mixture was then reacted with 1.9 μl of a 100 mMpeptide stock solution (in DMSO) for two hours at 25° C. on a rockingshaker. The reaction mixture was subsequently dialyzed 2×2 hours against2 liters of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C.

Immunization of Mice:

Female Balb/c mice were vaccinated with one of the two Der p I peptidescoupled to Qβ capsid protein without the addition of adjuvants. Two micefor each vaccine were used. 30 μg of total protein of each sample wasdiluted in PBS to 200 μl and injected subcutaneously on day 0 and day14. Mice were bled retroorbitally on day 21 and their serum was analyzedusing a Der p I peptide-specific ELISA.

ELISA

The Der p I peptides “Der p I p52” and “Der p I p117” were individuallycoupled to bovine RNAse A using the chemical cross-linker sulfo-SPDP.ELISA plates were coated with coupled RNAse preparations at aconcentration of 10 mg/ml. The plates were blocked and then incubatedwith serially diluted mouse sera. Bound antibodies were detected withenzymatically labeled anti-mouse IgG antibody. As a control, preimmunesera of the same mice were also tested. Control ELISA experiments usingsera from mice immunized with unrelated peptides crosslinked to Qβ orother carriers showed that the antibodies detected were specific for therespective peptide. The results are shown in FIGS. 9A and 9B.

FIG. 9A and FIG. 9B show ELISA analyses of IgG antibodies specific for“Der p I p52” (FIG. 9A) and specific for “Der p I p117” (FIG. 9B) insera of mice immunized against the Der p I peptides coupled to Qβ capsidprotein. “p52” and “p117”, as used in FIGS. 9A and 9B, stand for thevaccine injected in the mice, from which the sera are derived.

As a control, pre-immune sera from the same mice were analyzed (day 0).Results for indicated serum dilutions are shown as optical density at450 nm. On day 21, all vaccinated mice made specific IgG antibodiesagainst the Der p I peptide they were vaccinated with but not againstthe other Der p I peptide. No Der p I peptide-specific antibodies weredetected before vaccination (day 0).

Both Der p I peptide vaccines were highly immunogenic in the absence ofadjuvants. All vaccinated mice made good antibody responses specific forthe peptide in the vaccine preparation.

Example 46 Coupling of Der p 1 Peptides to HBcAg-149-Lys-2Cys-Mut, i.e.Cys-Free HBcAg

The following peptides derived from the house dust mite allergen Der p 1were chemically synthesized: Der p I p52 (aa 52-72, with an additionalcysteine-glycine linker at the N terminus): CGNQSLDLAEQELVDCASQHGCH, Derp I p117 (aa 117-137): CQIYPPNANKIREALAQTHSA. These peptides are usedfor chemical coupling to HBcAg-149-lys-2cys-Mut, i.e. cys-free HBcAg.

A solution of 1.25 ml of 0.8 mg/ml HBcAg-149-lys-2cys-Mut capsid protein(Example 31) in PBS, pH 7.4 is reacted for 30 minutes with 93 μl of asolution of 13 mg/ml Sulfo-MBS (Pierce) in H₂O at 25° C. on a rockingshaker. The reaction solution is subsequently dialyzed overnight against2 L of 20 mM Hepes, 150 mM NaCl, pH 7.4. After buffer exchange thereaction solution is dialyzed for another 2 hours. The dialyzed reactionmixture is then reacted with 1.8 μl of a 100 mM peptide stock solution(in DMSO) for 2 hours at 25° C. on a rocking shaker. The reactionmixture is subsequently dialyzed against 2 liters of 20 mM Hepes, 150 mMNaCl, ph 7.4 overnight at 4° C. followed by buffer exchange and another2 hours of dialysis.

Example 47 Coupling of Der p I Peptides to Type-1 Pili of E. coli

The following peptides derived from the house dust mite allergen Der p Iwere chemically synthesized: Der p I p52 (aa 52-72, with an additionalcysteine-glycine linker at the N terminus) and CGNQSLDLAEQELVDCASQHGCH,Der p I p117 (aa 117-137): CQTYPPNANKIREALAQTHSA. These peptides areused for chemical coupling to Type-1 pili of E. coli.

A solution of 400 μl of 2.5 mg/ml Type-1 pili of E. coli in 20 mM Hepes,pH 7.4, is reacted for 60 minutes with 60 μl of a 100 mM Sulfo-MBS(Pierce) solution in (H₂O) at RT on a rocking shaker. The reactionmixture is desalted on a PD-10 column (Amersham-Pharmacia Biotech), Theprotein-containing fractions eluating from the column are pooled(containing approximately 1 mg protein, i.e. derivatized pili) andreacted with a three-fold molar excess of peptide. For example, to 500ul eluate containing approximately 1 mg derivatized pili, 2.34 ul of a100 mM peptide stock solution (in DMSO) is added. The mixture isincubated for four hours at 25° C. on a rocking shaker and subsequentlydialyzed against 2 liters of 20 mM Hepes, 150 mM NaCl, pH 7.2 overnightat 4° C.

Example 48 Coupling of HumanVEGFR-II Peptide to Type-1 Pili of E. coliand Immunization of Mice with Vaccines Comprising Type-1Pili-HumanVEGFR-II Peptide Arrays

Coupling of humanVEGFR-II Peptide to Type-1 Pili of E. coli

The human VEGFR II peptide with the sequence CTARTELNVGIDFNWEYPSSKHQHKKwas chemically synthesized and used for chemical coupling to Type-1 piliof E. coli.

A solution of 1400 μl of 1 mg/ml pili protein in 20 mM Hepes, pH 7.4,was reacted for 60 minutes with 85 μl of a 100 mM Sulfo-MBS (Pierce)solution in (H₂O) at RT on a rocking shaker. The reaction mixture wasdesalted on a PD-10 column (Amersham-Pharmacia Biotech). Theprotein-containing fractions eluting from the column were pooled(containing approximately 1.4 mg protein) and reacted with a 2.5-foldmolar excess (final volume) of human VEGFR II peptide. For example, to200 μl eluate containing approximately 0.2 mg derivatized pili, 2.4 μlof a 10 mM peptide solution (in DMSO) was added. The mixture wasincubated for four hours at 25° C. on a rocking shaker and subsequentlydialyzed against 2 liters of 20 mM Hepes, pH 7.2 overnight at 4° C.

Immunization of Mice

Female C3H-HeJ (Toll-like receptor 4 deficient, LPS non-responder mice)and C3H-HeN (wild-type) mice were vaccinated with the human VEGFR-IIpeptide coupled to Type-1 pili protein without the addition ofadjuvants. Approximately 100 μg of total protein of each sample wasdiluted in PBS to 200 μl and injected subcutaneously on day 0, day 14and day 28. Mice were bled retroorbitally on day 14, 28 and day 42 andserum of day 42 was analyzed using a human VEGFR-II specific ELISA

ELISA

Sera of immunized mice were tested in ELISA with immobilized humanVEGFR-II peptide and the extracellular domain of the human VEGFR-II (R&DSystems GmbH, Wiesbaden).

Human VEGFR-II peptide was coupled to bovine RNAse A using the chemicalcross-linker sulfo-SPDP. ELISA plates were coated with coupled RNAse Aat a concentration of 10 μg/ml. The human extracellular domain ofVEGFR-II was adsorbed to the plates at a concentration of 2 μg/ml. Theplates were blocked and then incubated with serially diluted mouse sera.Bound antibodies were detected with enzymatically labeled anti-mouse IgGantibody. As a control, preimmune sera of the same mice were alsotested. Control ELISA experiments using sera from mice immunized withuncoupled carrier showed that the antibodies detected were specific forthe respective peptide. The results for human VEGFR II peptide coupledto Type-1 pili are shown in FIG. 10. In particular, FIG. 10A. and FIG.10B show ELISA analyses of IgG antibodies specific for human VEGFR IIpeptide and extracellular domain of human VEGFR II, respectively, insera of mice immunized against human VEGFR II peptide and theextracellular domain of human VEGFR II each coupled to Type-1 piliprotein.

Female C3H-HeJ (Toll-like receptor 4 deficient, LPS-nonresponder) andC3H-HeN (wild-type) mice were vaccinated subcutaneously with 100 ug ofvaccine in PBS on day 0, 14 and 28. Serum IgG against the peptide(coupled to RNAse A) and the extracellular domain of human VEGFR II weremeasured on day 42. As a control, preimmune sera from the same mice wereanalyzed. Results for indicated serum dilutions are shown as opticaldensity at 450 nm. The average of three mice each (including standarddeviations) are shown. All vaccinated mice made high IgG antibody titersagainst the human VEGFR-II peptide as well as the extracellular domainof human VEGFR-II (KDR) and no difference was noted between micedeficient for the Toll-like receptor 4 and wild-type mice. The latter isremarkable since it demonstrates that formation of high IgG antibodytiters against the human VEGFR-II peptide as well as the extracellulardomain of human VEGFR-II is independent of endotoxin contaminations.

Example 49 Coupling of HumanVEGFR-II Peptide to Qβ Capsid Protein andImmunization of Mice with Vaccines Comprising Qβ CapsidProtein-HumanVEGFR-II Peptide Arrays

Coupling of HumanVEGFR-II Peptide to Qβ Capsid Protein

The human VEGFR II peptide with the sequence CTARTELNVGIDFNWEYPSSKHQHKKwas chemically synthesized and is used for chemical coupling to Qβcapsid protein.

A solution of 1 ml of 1 mg/ml Qβ capsid protein in 20 mM Hepes, 150 mMNaCl pH 7.4 was reacted for 45 minutes with 20 μl of 100 mM Sulfo-MBS(Pierce) solution in (H₂O) at RT on a rocking shaker. The reactionsolution was subsequently dialyzed twice for 2 hours in 2 L of 20 mMHepes, pH 7.4 at 4° C. 1000 μl of the dialyzed reaction mixture was thenreacted with 12 μl of a 10 mM human VEGFR II peptide solution (in DMSO)for four hours at 25° C. on a rocking shaker. The reaction mixture wassubsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, pH 7.4at 4° C.

1 ml of a solution consisting of 2 mg/ml Qβ capsid protein in 20 mMHepes, 150 mM NaCl, pH 7.4 was reacted for 30 minutes with 102 μl of asolution of 13 mg/ml Sulfo-MBS (Pierce) in H₂O at 25° C. on a rockingshaker. The reaction solution was subsequently dialyzed twice for 2hours against 2 L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. 440 μl ofthe dialyzed reaction mixture was then reacted with 1.9 μl of a 100 mMpeptide stock solution (in DMSO) for two hours at 25° C. on a rockingshaker. The reaction mixture was subsequently dialyzed 2×2 hours against2 liters of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C.

Immunization of Mice

C57BL/6 mice are vaccinated with the human VEGFR-II peptide coupled toQβ protein without the addition of adjuvants. Approximately 50 μg oftotal protein of each sample is diluted in PBS to 200 ul and injectedsubcutaneously on day 0, day 14 and day 28. Mice are bled retroorbitallyon day 14, 28 and day 42 and serum of day 42 is analyzed using a humanVEGFR-II specific ELISA

Example 50 Coupling of HumanVEGFR-II Peptide to HBcAg-149-Lys-2Cys-MutCapsid Protein, i.e. Cys-Free HBcAg, and Immunization of Mice withVaccines Comprising HBcAg-149-Lys-2Cys-Mut Capsid Protein-HumanVEGFR-IIPeptide Arrays

Coupling of HumanVEGFR-II Peptide to HBcAg-149-Lys-2Cys-Mut CapsidProtein

The human VEGFR II peptide with the sequence CTARTELNVGIDFNWEYPSSKHQHKKwas chemically synthesized and is used for chemical coupling toHBcAg-149-lys-2cys-Mut capsid protein.

A solution of 3 ml of 0.9 mg/ml cys-free HbcAg capsid protein (cf.Example 31) in PBS, pH 7.4 is reacted for 45 minutes with 37.5 μl of 100mM Sulfo-MBS (Pierce) solution in (H₂O) at RT on a rocking shaker. Thereaction solution is subsequently dialyzed overnight against 2 L of 20mM Hepes, pH 7.4. After buffer exchange the reaction solution isdialyzed for another 2 hours. The dialyzed reaction mixture is thenreacted with 3 μl of a 10 mM human VEGFR II peptide solution (in DMSO)for 4 hours at 25° C. on a rocking shaker. The reaction mixture issubsequently dialyzed against 2 liters of 20 mM Hepes, pH 7.4 overnightat 4° C. followed by buffer exchange and another 2 hours of dialysis.

Example 51 Construction of HBcAg1-183Lys

Hepatitis core Antigen (HBcAg) 1-183 was modified as described inExample 23. A part of the c/el epitope (residues 72 to 88) region(Proline 79 and Alanine 80) was genetically replaced by the peptideGly-Gly-Lys-Gly-Gly (HBcAg1-183Lys construct). The introduced Lysineresidue contains a reactive amino group in its side chain that can beused for intermolecular chemical crosslinking of HBcAg particles withany antigen containing a free cysteine group. PCR methods essentially asdescribed in Example 1 and conventional cloning techniques were used toprepare the HBcAg1-183Lys gene.

The Gly-Gly-Lys-Gly-Gly sequence was inserted by amplifying two separatefragments of the HBcAg gene from pEco63, as described above in Example23 and subsequently fusing the two fragments by PCR to assemble the fulllength gene. The following PCR primer combinations were used:

fragment 1:

-   -   Primer 1: EcoRIHBcAg(s) (see Example 23)    -   Primer 2: Lys-HBcAg(as) (see Example 23)

fragment 2:

-   -   Primer 3: Lys-HBcAg(s) (see Example 23)    -   Primer 4: HBcAgwtHindIIII

CGCGTCCCAAGCTTCTAACATTGAGATTCCCGAGATTG

-   -   -   Assembly:

    -   Primer 1: EcoRIHBcAg(s) (see example 23)

    -   Primer 2: HBcAgwtHindIIII

The assembled full length gene was then digested with the EcoRI (GAATTC)and HindIII (AAGCTT) enzymes and cloned into the pKK vector (Pharmacia)cut at the same restriction sites.

Example 52 Coupling of muTNFa Peptide to HBcAg1-183Lys and Immunizationof Mice with Vaccines Comprising HBcAg1-183Lys-muTNFa Peptide Arrays

A. Coupling of muTNFa Peptide to HBcAg1-183Lys

HBcAg1-183Lys at a concentration of 0.6 mg/ml (29 μM) was treated withiodacetamide as described in Example 32. HBcAg1-183Lys was then reactedwith a fifty-fold excess of the cross-linker Sulfo-MBS, as described inExample 32, and dialyzed overnight against 20 mM Hepes, pH 7.2, at 4° C.Activated (derivatized) HBcAg1-183Lys was reacted with a five-fold molarexcess of the peptide muTNFa (sequence: CGGVEEQLEWLSQR, diluted directlyinto the HBcAg1-183Lys solution from a 100 mM stock solution in DMSO) atRT for 4 hours. The coupling reaction (about 1 ml solution) was dialyzedagainst 2×2 liters of 20 mM HEPES pH 7.2, at 4° C., for 4 hours. Thedialyzed coupling reaction was frozen in aliquots in liquid nitrogen andstored at −80° C. until immunization of the mice.

Immunization

Two mice (female Balb/c) were immunized intravenously at day 0 and 14with 100 μg HBcAg1-183Lys coupled to the muTNFa peptide, per animal,without adjuvant. Antibodies specific for the muTNFa peptide (coated asa Ribonuclease A conjugate) and for native TNFα protein (Sigma) in theserum were determined at day 21 by ELISA.

ELISA

Murine TNFα protein (Sigma) was coated at a concentration of 2 μg/ml. Asa control, preimmune sera from the same mice used for immunization weretested. FIG. 14 shows the result of the ELISA experiment, demonstratingthat immunization with HBcAg1-183Lys coupled to the muTNFa peptide (Fulllength HBc-TNF) generated an immune response specific for the murineTNFα protein. The sera from mice bled on day 0 (preimmune) and 21 weretested at three different dilutions. Each bar is the average of thesignal obtained with sera from two mice. Thus, vaccination withHBcAg1-183Lys coupled to the muTNFa peptide induced an immune responseagainst a self-antigen, since the amino acid sequence of the muTNFapeptide is derived from the sequence of mouse TNFα protein.

Example 53 Coupling of 3′TNF II Peptide to 2cysLys-mut HBcAg1-149 andImmunization of Mice with Vaccines Comprising 2cysLys-mut HBcAg1-1493′TNF II Peptide Arrays

Coupling of the 3′TNF II Peptide to 2cysLys-Mut HBcAg1-149

2cysLys-mut HBcAg1-149 was reacted at a concentration of 2 mg/ml for 30min. at RT with a fifty-fold excess of cross-linker in 20 mM Hepes, 150mM NaCl, pH 7.2. Excess cross-linker was removed by dialysis overnight,and activated (derivatized) 2cysLys-mut HBcAg1-149 capsid protein wasreacted with a ten-fold excess of 3′TNF II peptide (SEQ:SSQNSSDKPVAHVVANHGVGGC, diluted from a 100 mM stock solution in DMSO)for 4 hours at RT. The reaction mixture was then dialyzed overnight in adialysis tubing with a molecular weight cutoff of 50000 Da, frozen inliquid nitrogen and stored at −80° C. until immunization of the mice.

Immunization of Mice

3 Female C3H/HeN mice, 8 weeks of age were vaccinated with the 3′TNF IIpeptide coupled to 2cysLys-mut HBcAg1-149 without the addition ofadjuvants. 50 μg of total protein was diluted in PBS to 200 μl andinjected subcutaneously (100 μl on two inguinal sides) on day 0 and day14. Mice were bled retroorbitally on day 0 and 21, and their serum wereanalyzed in an ELISA specific for murine TNFα protein.

ELISA

Murine TNFα protein (Sigma) was coated at a concentration of 2 μg/ml. Asa control, preimmune sera from the same mice used for immunization weretested. FIG. 15 shows the result of the ELISA, demonstrating thatimmunization with 2cysLys-mut HBcAg1-149 coupled to the 3′TNF II peptidegenerated an immune response specific for the murine TNFα protein. Thesera from mice bled on day 0 (preimmune) and 21 were tested at threedifferent dilutions. Each bar is the average of the signal obtained withsera from 3 mice. Thus, vaccination with 2cysLys-mut HBcAg1-149 coupledto the 3′TNF II peptide induced an immune response specific for aself-antigen, since the amino acid sequence of the 3′TNF II peptide isderived from the sequence of murine TNFα protein.

Example 54 Coupling of AP 1-15, Aβ 1-27 and Aβ 33-42 Peptides to Qβ andImmunization of Mice with Vaccines Comprising Qβ-Aβ Peptide Arrays A.Coupling of Aβ 1-15 and Aβ 33-42 Peptides to Qβ Capsid Protein Using theCross-Linker SMPH.

The following Aβ peptides were chemically synthesized:DAEFRHDSGYEVHHQGGC (abbreviated as “Aβ 1-15”), a peptide which comprisesthe amino acid sequence from residue 1-15 of human Aβ, fused at itsC-terminus to the sequence GGC for coupling to Qβ capsid protein andCGHGNKSGLMVGGVVIA (abbreviated as “Aβ 33-42”) a peptide which comprisesthe amino acid sequence from residue 33-42 of Aβ, fused at itsN-terminus to the sequence CGHGNKS for coupling to Qβ capsid protein.Both peptides were used for chemical coupling to Qβ as described in thefollowing.

A solution of 1.5 ml of 2 mg/ml Qβ capsid protein in 20 mM Hepes 150 mMNaCl pH 7.4 was reacted for 30 minutes with 16.6 μl of a solution of 65mM SMPH (Pierce) in H₂O, at 25° C. on a rocking shaker. The reactionsolution was subsequently dialyzed twice for 2 hours against 2 L of 20mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. in a dialysis tubing withMolecular Weight cutoff 10000 Da. 450 μl of the dialyzed reactionmixture, which contains activated (derivatized) Qβ, was then reactedwith 6.5 μl of each of the corresponding 50 mM peptide stock solution(in DMSO) for two hours at 15° C. on a rocking shaker. 200 μl of thereaction mixture was subsequently dialyzed overnight against 2 liters of20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C., and the next morning foranother two hours after change of buffer. The reaction mixture was thenfrozen in aliquots in liquid Nitrogen and stored at −80° C. untilimmunization of the mice.

The results of the coupling experiments were analyzed by SDS-PAGE, andare shown in FIG. 13A and FIG. 13B. The arrows point to the bandcorresponding to one, respectively two peptides coupled to one Qβsubunit (FIG. 13A), or one peptide coupled to one Qβ subunit (FIG. 13B).Molecular weights of marker proteins are given on the left margin ofFIG. 13A and FIG. 13B.

The samples loaded on the gel of FIG. 13A are the following:

1: derivatized Qβ; 2: Qβ coupled with “Aβ1-15”, supernatant of thesample taken at the end of the coupling reaction, and centrifuged; 3: Qβcoupled with “Aβ1-15”, pellet of the sample taken at the end of thecoupling reaction, and centrifuged. 4: Qβ coupled with “Aβ1-15”,supernatant of a sample left to stand 24 hours at 4° C., undialyzed andcentrifuged. 5: Qβ coupled with “Aβ1-15”, pellet of a sample left tostand 24 hours at 4° C., undialyzed and centrifuged.6: Qβ coupled with “Aβ1-15”, supernatant of the sample taken afterdialysis of the coupling reaction, and centrifuged.

The samples loaded on the gel of FIG. 13B are the following: 1:derivatized Qβ 2: Qβ coupled with “Aβ33-42”, supernatant of the sampletaken at the end of the coupling reaction, and centrifuged. 3: Qβcoupled with “Aβ33-42”, pellet of the sample taken at the end of thecoupling reaction, and centrifuged. 4: Qβ coupled with “Aβ33-42”,supernatant of a sample left to stand 24 hours at 4° C., undialyzed andcentrifuged. 5: Qβ coupled with “Aβ33-42”, pellet of a sample left tostand 24 hours at 4° C., undialyzed and centrifuged.

6: Qβ coupled with “Aβ33-42”, supernatant of the sample taken afterdialysis of the coupling reaction, and centrifuged.

B. Coupling of “Aβ1-27” Peptide to Qβ Capsid Protein Using theCross-Linker SMPH.

The following Aβ peptide (“Aβ1-27”) was chemically synthesizedDAEFRHDSGYEVHHQKLVFFAEDVGSNGGC. This peptide comprises the amino acidsequence from residue 1-27 of human Aβ, fused at its C-terminus to thesequence GGC for coupling to Qβ capsid protein.

A first batch of “Aβ1-27” coupled to Qβ capsid protein, in the followingabbreviated as “Qβ-Aβ1-27 batch 1” was prepared as follows:

A solution of 1.5 ml of 2 mg/ml Qβ capsid protein in 20 mM Hepes 150 mMNaCl pH 7.4 was reacted for 30 minutes with 16.6 μl of a solution of 65mM SMPH (Pierce) in H₂O, at 25° C. on a rocking shaker. The reactionsolution was subsequently dialyzed twice for 2 hours against 2 L of 20mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. in a dialysis tubing withMolecular Weight cutoff 10000 Da. 450 μl of the dialyzed reactionmixture was then reacted with 6.5 μl of a 50 mM peptide stock solution(in DMSO) for two hours at 15° C. on a rocking shaker. 200 μl of thesample was then aliquoted, frozen in liquid Nitrogen and stored at −80°C. until immunization of the mice.

A second batch of “Aβ 1-27” coupled to Qβ capsid protein, in thefollowing abbreviated as “Qβ/Aβ1-27 batch 2” was prepared as follows:

500 μl of Qβ capsid protein in 20 mM Hepes 150 mM NaCl pH 7.4 wasreacted for 30 minutes with 11.3 μl of a solution of 32.5 mM SMPH(Pierce) in H₂O, at 25° C. on a rocking shaker. The reaction solutionwas subsequently dialyzed twice for 2 hours against 2 L of 20 mM Hepes,150 mM NaCl, pH 7.4 at 4° C. in a dialysis tubing with Molecular Weightcutoff 3500 Da (SnakeSkin, Pierce). The dialyzed reaction mixture wasthen reacted with 3.6 μl of a 50 mM peptide stock solution (in DMSO) fortwo hours at 15° C. on a rocking shaker. The reaction mixture was thendialyzed 2× against 1 l 20 mM Hepes, 150 mM NaCl, pH 7.4 for 1 hour andovernight after a last change of buffer, using a dialysis membrane witha 50000 Da cutoff (Spectrapor, spectrum). The reaction mixture was thenfrozen in aliquots in liquid nitrogen and stored at −80° C. untilimmunization of the mice. “Qβ-Aβ 1-27 batch 1” was used for the firstimmunization, while “Qβ-Aβ 1-27 batch 2” was used for the boost.

The result of the coupling experiment was analyzed by SDS-PAGE, and isshown in FIG. 13C. The arrow points to the band corresponding to onepeptide coupled to one Qβ subunit.

The samples loaded on the gel of FIG. 13C are the following:

M: protein marker. 1: Qβ capsid protein 2: derivatized Qβ, supernatantof the sample taken at the end of the derivatization reaction, andcentrifuged. 3: derivatized Qβ, pellet of the sample taken at the end ofthe derivatization reaction, and centrifuged. 4: Qβ coupled with“Aβ1-27”, supernatant of the sample taken at the end of the couplingreaction, and centrifuged. 5: Qβ coupled with “Aβ1-27”, pellet of thesample taken at the end of the coupling reaction, and centrifuged. 6: Qβcoupled with “Aβ1-27”, supernatant of the sample taken after dialysis ofthe coupling reaction, and centrifuged. 7: Qβ coupled with “Aβ1-27”,pellet of the sample taken after dialysis of the coupling reaction, andcentrifuged.

C. Coupling of “Aβ 1-15” Peptide to Qβ Capsid Protein Using theCross-Linker Sulfo-GMBS

A solution of 500 μl of 2 mg/ml Qβ capsid protein in 20 mM Hepes 150 mMNaCl pH 7.4 was reacted for 30 minutes with 5.5 μl of a solution of 65mM SMPH (Pierce) in H₂O, at 25° C. on a rocking shaker. The reactionsolution was subsequently dialyzed twice for 2 hours against 2 L of 20mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. in a dialysis tubing withMolecular Weight cutoff 10 000 Da. 500 μl of the dialyzed reactionmixture was then reacted with 6.5 μl of the 50 mM peptide stock solution(in DMSO) for two hours at 15° C. on a rocking shaker. 200 μl of thereaction mixture was subsequently dialyzed overnight against 2 liters of20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C., and the next morning foranother two hours after change of buffer. The reaction mixture was thenfrozen in aliquots in liquid Nitrogen and stored at −80° C. untilimmunization of the mice.

The result of the coupling experiment was analyzed by SDS-PAGE, and isshown in FIG. 13D. The arrow points to the band corresponding to one,two and three peptides, respectively, coupled to one Qβ subunit.

The samples loaded on the gel of FIG. 13D are the following:

M: protein marker. 1: derivatized Qβ 2: Qβ coupled with “Aβ1-15”,supernatant of the sample taken at the end of the coupling reaction, andcentrifuged. 3: Qβ coupled with “Aβ1-15”, pellet of the sample taken atthe end of the coupling reaction, and centrifuged. 4: Qβ coupled with“Aβ1-15”, supernatant of a sample left to stand 24 hours at 4° C.,undialyzed and centrifuged. 5: Qβ coupled with “Aβ1-15”, pellet of asample left to stand 24 hours at 4° C., undialyzed and centrifuged. 6:Qβ coupled with “Aβ1-15”, supernatant of the sample taken after dialysisof the coupling reaction, and centrifuged.

D. Coupling of “Aβ 1-15” to Qβ Capsid Protein Using the Cross-LinkerSulfo-MBS.

500 μl of Qβ capsid protein in 20 mM Hepes 150 mM NaCl pH 7.4 wasreacted for 30 minutes with 14.7 μl of a solution of 100 mM Sulfo-MBS(Pierce) in H₂O, at 25° C. on a rocking shaker. The reaction solutionwas subsequently dialyzed twice for 2 hours against 2 L of 20 mM Hepes,150 mM NaCl, pH 7.4 at 4° C. in a dialysis tubing (SnakeSkin, Pierce)with Molecular Weight cutoff 3500 Da. The dialyzed reaction mixture wasthen reacted with 7.2 μl of a 50 mM peptide stock solution (in DMSO) fortwo hours at 15° C. on a rocking shaker. The reaction mixture was thendialyzed 3× over 4 hours against 2 l 20 mM Hepes, 150 mM NaCl, pH 7.4using a dialysis membrane with a 50000 Da cutoff (Spectrapor, spectrum).The reaction mixture was then frozen in aliquots in liquid nitrogen andstored at −80° C. until immunization of the mice.

The result of the coupling experiment was analyzed by SDS-PAGE, and isshown in FIG. 13E. The arrow points to the band corresponding to onepeptide coupled to one Qβ subunit.

The samples loaded on the gel of FIG. 13E are the following:

1: Qβ capsid protein 2: derivatized Qβ, supernatant of the sample takenat the end of the derivatization reaction, and centrifuged. 3:derivatized Qβ, pellet of the sample taken at the end of thederivatization reaction, and centrifuged. 4: derivatized Qβ, supernatantof the sample taken at the end of the dialysis of the derivatizationreaction, and centrifuged. 5: derivatized Qβ, pellet of the sample takenat the end of the dialysis of the derivatization reaction, andcentrifuged. 6: Qβ coupled with “Aβ1-15”, supernatant of the sampletaken at the end of the coupling reaction, and centrifuged. 7: Qβcoupled with “Aβ1-15”, pellet of the sample taken at the end of thecoupling reaction, and centrifuged. 8: Qβ coupled with “Aβ1-15”,supernatant of the sample taken after dialysis of the coupling reaction,and centrifuged.

E. Immunization of Mice:

Five groups of female C57BL/6 mice, three mice per group, 8 weeks of agewere vaccinated each with one of the five Aβ peptide-Qβ capsid proteinconjugates without the addition of adjuvant. 25 μg of total protein ofeach sample was diluted in PBS to 200 μl and injected subcutaneously onday 0 and day 14. Mice were bled retroorbitally on day 0 (preimmune) and21 and their serum was analyzed in an ELISA. “Aβ 1-15” peptide wascoupled to Qβ with three different cross-linkers, resulting in threedifferent vaccine preparations (“Qb-Aβ1-15 SMPH”, “Qβ-Ab1-15 SMBS”,“Qb-Aβ1-15 SGMBS”; see ELISA section for the results).

F. ELISA

All three Aβ peptides were individually coupled to bovine RNAse A usingthe chemical cross-linker SPDP as follows: a solution of 10 mg RNAse Ain 2 mL PBS (50 mM Phosphate buffer, 150 mM NaCl pH 7.2) was reactedwith 100 μl of a 20 mM SPDP solution in DMSO, at 25° C. for 60 min. on arocking shaker. Excess cross-linker was separated from activated(derivatized) RNAse A by gel filtration using a PD 10 column(Pharmacia). The protein containing fractions were pooled andconcentrated to a volume of 2 ml using centrifugal filters (5000 MWCO).A sample of 333 μl of the derivatized RNAse A solution was reacted with2 μl of the peptide stock solution (50 mM in DMSO). The couplingreaction was followed spectrophotometrically.

ELISA plates were coated with RNAse A coupled to peptide at aconcentration of 10 μg/ml. The plates were blocked and then incubatedwith serially diluted mouse sera. Bound antibodies were detected withenzymatically labeled anti-mouse IgG antibody. Preimmune sera or controlsera from mice immunized with unrelated peptides conjugated to Qβ,showed that the antibodies detected were specific for the respectivepeptide. FIG. 14A, FIG. 14B and FIG. 14C, respectively, show ELISAanalyses of IgG antibodies specific for “Aβ 1-15”, “Aβ 1-27” and “Aβ33-42”, respectively, in sera of mice immunized against “Aβ 1-15”, “Aβ1-27” and “Aβ 33-42”, respectively, coupled to Qβ capsid protein. Thedenominations on the abscissa stand for the vaccine injected in the micefrom which the sera are derived, and describe the peptide and thecross-linker used to make the respective vaccine. All sera were measuredagainst the three peptides coupled to RNAse A, and the results show thatwhile there is cross-reactivity between the antibodies raised againstpeptide 1-15 and 1-27, no such cross reactivity is observed againstpeptide 33-42, demonstrating the specificity of the immune response.Likewise, The ELISA titers obtained, expressed as the dilution of theserum yielding an ELISA signal three standard deviations abovebackground, were very high, and ranged from 60′000 to 600′000. No Aβpeptide-specific antibodies were detected in the controls (pre-immunemice).

Example 55 Introduction of Cys-Containing Linkers, Expression, andPurification of Anti-Idiotypic IgE Mimobodies and their Coupling to QβCapsid Protein A. Construction of Plasmids for the Expression ofMimobodies for Coupling to Qβ Capsid Protein

Plasmids were based on the expression plasmid VAE051-pASK116. Thisplasmid contains the coding regions for the heavy chain and for thelight chain of the mimobody. The following primers were used tointroduce cys-containing linkers at the C-terminus of the heavy chain:

Primer CA2F: CGGCTCGAGCATCACCATCACCATCACGGTGAAGTTAAACTGCAGCTG GAGTCGPrimer CA1R: CATGCCATGGTTAACCACAGGTGTGGGTTTTATCACAAGATTTGGGCT CAACPrimer CB1R: CATGCCATGGTTAACCACACGGCGGAGAGGTGTGGGTTTTATCACAAGATTTGGGCTCAAC Primer CC1R:CCAGAAGAACCCGGCGGGGTAGACGGTTTCGGGCTAGCACAAGATTT GGGCTCAACTC Primer CC1F:CGCCGGGTTCTTCTGGTGGTGCTCCGGGTGGTTGCGGTTAACCATGGA GAAAATAAAGTGPrimer CCR2: CTCCCGGGTAGAAGTCACA.1. Construction of pCA2:

Primers CA2F and CA1R were used to amplify a 741 bp fragment encodingpart of the heavy chain with an extension encoding the cys-containinglinker sequence. VAE-pASK116 served as template for the Pfx polymerase(Roche) in the PCR cycler (Robo) at (initial denaturation at 92° C.,cycling: 92° C., 30 s; 48° C., 30 s; 68° C., 60s) for 5 cycles followedby 30 cycles with 92° C., 30 s; 58° C., 30 s; 68° C., 1 min. The PCRproduct of the appropriate size was purified using the Qiagen PCRpurification kit and digested with XhoI and NcoI according to therecommendation of the manufacturer (Gibco). The product was purifiedfrom an agarose gel with the Qiagen gel extraction kit. PlasmidVAE-pASK116 was in parallel cleaved with XhoI and NcoI and a 3.7 kb bandpurified from agarose gels. Appropriate aliquots of the XhoI-NcoIdigested PCR product and the plasmid were ligated overnight at 16° C.using T4 DNA ligase according to the manufacturer's protocol (Gibco).The ligation product was transformed into competent E. coli XL-1 cellswhich were plated on agarose plates containing chloramphenicol. Singlecolonies were expanded in LB/chloramphenicol medium, plasmid wasprepared (Qiagen mini plasmid kit) and tested for the presence of theappropriate XhoI-NcoI insert size after digestion with the correspondingenzymes. A correspondingly positive plasmid termed pCA2 was sent forsequencing on both strands which confirmed the identity of the plasmidincluding the cys-containing linker.

A.2. Construction of pCB2:

Primers CA2F and CB1R were used to introduce linker 2 at the 5′ end ofthe heavy chain coding sequence and the same conditions as described insection A1. The resulting PCR product was 750 bp and cloned intoVAE051-pASK116 as described in section A.1.

A.3. Construction of pCC2:

Plasmid pCC2 was constructed in a two step procedure: A first PCRproduct of 754 bp was amplified using primers CA2F and CC1R. A secondPCR product of 560 bp was produced using primers CC1F and CC2R. For bothPCRs VAE051-pASK116 was used as template and conditions were asdescribed in section A1. Both PCR products were isolated from agarosegels, mixed with primers CA2F and CC2R and a third PCR was performedthat resulted in a 1298 bp fragment. This fragment was isolated anddigested with XhoI and NcoI. The resulting 780 bp fragment was clonedinto VAE-pASK100 as described in section A.1.

B. Expression of Mimobodies

Competent E. coli W3110 cells were transformed with plasmids pCA2, pCB2and pCC2. Single colonies from chloramphenical agarose plates wereexpanded in liquid culture (LB+15 μg/ml chloramphenicol) overnight at37° C. 1 l of TB medium was then inoculated 1:50 v/v with the overnightculture and grown to OD600=3 at 28° C. Expression was induced with 1mg/1 anhydrotetracyclin. Cells were harvested after overnight cultureand centrifuged at 6000 rpm. Periplasma was isolated from cell pelletsby incubation in lysis buffer supplemented with polymyxin B sulfate for2 h at 4° C. Spheroblasts were separated by centrifugation at 6000 rpm.The resulting supernatant contained the mimobody and was dialysedagainst 20 mM Tris, pH 8.0.

C. Purification of Mimobodies

The introduced his6-tag allowed the purification of mimobody pCA2 andpCB2 by chromatography on Ni-NTA fast flow (Qiagen) according therecommendations of the manufacturer. If necessary, a polishing step on aprotein G fast flow column (Amersham Pharmacia Biotech) followed.Mimobodies were eluted with 0.1 M glycine pH 2.7, immediatelyneutralized by addition of NaOH and dialysed against 20 mM Hepes, 150 mMNaCl, pH 7.2.

pCC2 was purified by affinity chromatography on protein G only. Puritywas analysed by SDS-PAGE.

The protein sequences of the mimobodies were translated from the cDNAsequences. N-terminal sequences were confirmed by Edman sequencing ofpCA2 and pCB2.

The sequence of the light chains of pCA2, pCB2 and pCC2 is the same andas follows:

DIELVVTQPASVSGSPGQSITISCTGTRSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLGVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSC QVTHEGSTVEKTVAPTECS

The sequence of the heavy chain of pCA2 is:

EVKLQLEHHHHHHGEVKLQLESGPGLVKPSETLSLTCTVSGGSISSGGYYWTWIRQRPGKGLEWIGYIYYSGSTSYNPSLKSRVTMSVDTSKNQFSLRLTSVTAADTAVYYCARERGETGLYYPYYYIDVWGTGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC G

The sequence of the heavy chain of pCB2 is:

EVKLQLEHHHHHHGEVKLQLESGPGLVKPSETLSLTCTVSGGSISSGGYYWTWIRQRPGKGLEWIGYIYYSGSTSYNPSLKSRVTMSVDTSKNQFSLRLTSVTAADTAVYYCARERGETGLYYPYYYIDVWGTGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTS PPCG

The sequence of the heavy chain of pCC2 is:

EVKLQLEHHHHHHGEVKLQLESGPGLVKPSETLSLTCTVSGGSISSGGYYWTWIRQRPGKGLEWIGYIYYSGSTSYNPSLKSRVTMSVDTSKNQFSLRLTSVTAADTAVYYCARERGETGLYYPYYYIDVWGTGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCASPKPS TPPGSSGGAPGGC

D. Coupling of Mimobodies to Qβ Capsid Protein

D.1. Coupling of Mimobody pCC2 to Qβ Capsid Protein:

A solution of 1.25 ml of 4.5 mg/ml Qβ capsid protein in 20 mM

Hepes, 150 mM NaCl pH 7.2 was reacted for 30 minutes with 40 μl of aSMPH solution (Pierce) (from a 100 mM stock solution dissolved in DMSO)at 25° C. on a rocking shaker. The reaction solution was subsequentlydialyzed twice for 2 hours against 2 l of 20 mM Hepes, 150 mM NaCl, pH7.2 at 4° C. 6 μl of the dialyzed reaction mixture was then reacted with30 μl of the pCC2 solution (2.88 mg/ml) for at 25° C. over night on arocking shaker.

The reaction products were analysed on 16% SDS-PAGE gels under reducingconditions. Gels were either stained with Coomassie Brilliant Blue orblotted onto nitrocellulose membranes. Membranes were blocked, incubatedwith a polyclonal rabbit anti-Qb antiserum (dilution 1:2000) or a mousemonoclonal anti-Fab-mAb (Jackson ImmunoResearch) (dilution 1:2000).Blots were subsequently incubated with horse radishperoxidase-conjugated goat anti-rabbit IgG or horse radishperoxidase-conjugated goat anti-mouse IgG (dilutions 1:7000),respectively

The results are shown in FIG. 13A. Coupling products and educts wereanalysed on 16% SDS-PAGE gels under reducing conditions. In FIG. 13A“pCC2” corresponds to the mimobody before coupling. “Qβ deriv” standsfor derivatized Qβ before coupling, “Qβ-pCC2” for the product of thecoupling reaction. Gels were either stained with Coomassie BrilliantBlue or blotted onto nitrocellulose membranes. Membranes were blocked,incubated with a polyclonal rabbit anti-Qβ antiserum (dilution 1:2000)or an mouse monoclonal anti-Fab-mAb (Jackson ImmunoResearch) (dilution1:2000). Blots were subsequently incubated with horse radishperoxidase-conjugated goat anti-rabbit IgG or horse radishperoxidase-conjugated goat anti-mouse IgG (dilutions 1:7000),respectively. Enhanced chemoluminescence (Amersham Pharmacia ELC kit)was used to visualize the immunoreactive bands. Molecular weights ofmarker proteins are given on the left margin.

A coupling product of about 40 kDa could be detected (FIG. 13A, arrows).Its reactivity with the anti-Qβ antiserum and the anti-Fab antibodyrecognizing the mimobody clearly demonstrated the covalent coupling ofthe mimobody to Qβ.

D.2. Coupling of Mimobodies pCA2 and pCB2 to Qβ Capsid Protein:

A solution of 1.25 ml of 4.5 mg/ml Qβ capsid protein in 20 mM Hepes, 150mM NaCl pH 7.4 was reacted for 30 minutes with 40 μl of a SMPH (Pierce)(from a 100 mM stock solution dissolved in DMSO) at 25° C. on a rockingshaker. The reaction solution was subsequently dialyzed twice for 2hours against 2 l of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. pCA2 (1.2mg/ml) was reduced with 20 mM TCEP for 30 min at 25° C., pCB2 (4.2mg/ml) with 50 mM mercaptoethylamine at 37° C. Both mimobodies were thendialyzed twice against 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C.Coupling was performed by adding 6 μl of derivatized Qβ to 30 μl ofmimobody at 25° C. over night on a rocking shaker.

The reaction products were analysed on 16% SDS-PAGE gels under reducingconditions. Gels were either stained with Coomassie Brilliant Blue orblotted onto nitrocellulose membranes. Membranes were blocked, incubatedwith a polyclonal rabbit anti-Qb antiserum (Cytos, dilution 1:2000) oran mouse monoclonal anti-his6-mAb (Qiagen) (dilution 1:5000). Blots weresubsequently incubated with horse radish peroxidase-conjugated goatanti-rabbit IgG or horse radish peroxidase-conjugated goat anti-mouseIgG (dilutions 1:5000), respectively.

The results are shown in FIG. 13B and FIG. 13C. Coupling products andeducts were analysed on 16% SDS-PAGE gels under reducing conditions. InFIG. 15A and FIG. 15B “pCA2” and “pCB2” corresponds to the mimobodiesbefore coupling. “Qb deriv” stands for derivatized Qβ before couplingand “Qβ-pCA2” and “Qβ-pCA2” for the products of the coupling reaction.Gels were either stained with Coomassie Brilliant Blue or blotted ontonitrocellulose membranes. Membranes were blocked, incubated with apolyclonal rabbit anti-Qb antiserum (dilution 1:2000) or an mousemonoclonal anti-his6-mAb (Qiagen) (dilution 1:5000). Blots weresubsequently incubated with horse radish peroxidase-conjugated goatanti-rabbit IgG or horse radish peroxidase-conjugated goat anti-mouseIgG (dilutions 1:5000), respectively. Enhanced chemoluminescence(Amersham Pharmacia ECL kit) was used to visualize the immunoreactivebands. Molecular weights of marker proteins are given on the leftmargin.

Coupling products of about 40 kDa could be detected for both the pCA2and the pCB2 coupling (FIG. 15A and FIG. 15B, arrows). Its reactivitywith the anti-Qβ antiserum and the anti-his6 antibody recognizing theheavy chain of the mimobody clearly demonstrated the covalent couplingof the mimobody to Qβ.

Example 56 Coupling of Flag Peptides to Wt and Mutant Qβ Capsid ProteinUsing the Cross-Linker Sulfo-GMBS

The Flag peptide, to which a CGG sequence was added N-terminally forcoupling, was chemically synthesized and had the following sequence:CGGDYKDDDDK. This peptide was used for chemical coupling to wt Qβ capsidprotein and the Qβ mutants capsid protein as described in the following.

E. Coupling of Flag Peptide to Qβ Capsid Protein

A solution of 100 ul of 2 mg/ml Qβ capsid protein in 20 mM Hepes. 150 mMNaCl pH 7.2 was reacted for 60 minutes with 7 μl of a solution of 65 mMSulfo-GMBS (Pierce) in H₂O at 25° C. on a rocking shaker. The reactionsolution was subsequently dialyzed twice for 2 hours against 2 L of 20mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. 100 μl of the dialyzed reactionmixture was then reacted with 0.58 μl of 100 mM Flag peptide stocksolution (in H₂O) for two hours at 25° C. on a rocking shaker. Thereaction mixture was subsequently dialyzed 2×2 hours against 2 liters of20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C.

B. Coupling of Flag Peptide to Qβ-240 Capsid Protein

A solution of 100 ul of 2 mg/ml Qβ-240 capsid protein in 20 mM Hepes.150 mM NaCl pH 7.2 was reacted for 60 minutes with 7 μl of a solution of65 mM Sulfo-GMBS (Pierce) in H₂O at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. 100 μl of the dialyzedreaction mixture was then reacted with 0.58 μl of 100 mM Flag peptidestock solution (in H₂O) for two hours at 25° C. on a rocking shaker. Thereaction mixture was subsequently dialyzed 2×2 hours against 2 liters of20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C.

C. Coupling of Flag Peptides to Qβ-250 Capsid Protein

A solution of 100 ul of 2 mg/ml Qβ-250 capsid protein in 20 mM Hepes.150 mM NaCl pH 7.4 was reacted for 60 minutes with 7 μl of a solution of65 mM Sulfo-GMBS (Pierce) in H₂O at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. 100 μl of the dialyzedreaction mixture was then reacted with 0.58 μl of 100 mM Flag peptidestock solution (in H₂O) for two hours at 25° C. on a rocking shaker. Thereaction mixture was subsequently dialyzed 2×2 hours against 2 liters of20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C.

D. Coupling of Flag Peptides to Qβ-259 Capsid Protein

A solution of 100 ul of 2 mg/ml Q13-259 capsid protein in 20 mM Hepes.150 mM NaCl pH 7.4 was reacted for 60 minutes with 7 μl of a solution of65 mM Sulfo-GMBS (Pierce) in H₂O at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. 100 μl of the dialyzedreaction mixture was then reacted with 0.58 μl of 100 mM Flag peptidestock solution (in H₂O) for two hours at 25° C. on a rocking shaker.

The reaction mixture was subsequently dialyzed 2×2 hours against 2liters of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C.

The results of the coupling reactions of the Qβ mutants 240, 250 and 259to Flag peptide analyzed by SDS-PAGE are shown in FIG. 22 A. The loadingpattern was the following:

1. Derivatized Qβ-240 2. Qβ-240 coupled to the Flag peptide 3.Derivatized Qβ-250 4. Qβ-250 coupled to the Flag peptide 5. DerivatizedQβ-259 6. Qβ-259 coupled to the Flag peptide 7. Derivatized wt Qβ 8. wtQβ coupled to the Flag peptide 9. Protein Marker.

Comparison of the derivatized reaction with the coupling reactions showsthat for all the mutants and wt, coupling bands corresponding to 1 and 2peptides per subunit are visible. The band corresponding to theuncoupled Qβ subunit is very weak, indicating that nearly all subunitshave reacted with at least one Flag peptide. For the Qβ-250 mutant andwt Qβ, a band corresponding to three peptides per subunit is visible.The ratio of the intensities of the band corresponding to two peptidesper subunit and the band corresponding to 1 peptide per subunit isstrongest for wt, with a ratio of 1:1. this ratio is still high for theQβ-250 mutant, while it is significantly weaker for the Qβ-240 mutantand weakest for the Qβ-259 mutant.

Example 57 Coupling of Flag Peptide to Qβ Capsid Protein Using theCross-Linker Sulfo-MBS

The Flag peptide, to which a CGG sequence was added N-terminally forcoupling, was chemically synthesized and had the following sequence:CGGDYKDDDDK. This peptide was used for chemical coupling to wt Qβ capsidprotein and the Qβ mutant capsid protein as described in the following.

F. Coupling of Flag Peptides to Qβ Capsid Protein

A solution of 100 ul of 2 mg/ml Qβ capsid protein in 20 mM Hepes. 150 mMNaCl pH 7.2 was reacted for 60 minutes with 7 μl of a solution of 65 mMSulfo-MBS (Pierce) in H₂O at 25° C. on a rocking shaker. The reactionsolution was subsequently dialyzed twice for 2 hours against 2 L of 20mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. 100 μl of the dialyzed reactionmixture was then reacted with 0.58 μl of 100 mM Flag peptide stocksolution (in H₂O) for two hours at 25° C. on a rocking shaker. Thereaction mixture was subsequently dialyzed 2×2 hours against 2 liters of20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C.

B. Coupling of Flag Peptide to Qβ-240 Capsid Protein

A solution of 100 ul of 2 mg/ml Qβ-240 capsid protein in 20 mM Hepes.150 mM NaCl pH 7.2 was reacted for 60 minutes with 7 μl of a solution of65 mM Sulfo-MBS (Pierce) in H₂O at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. 100 μl of the dialyzedreaction mixture was then reacted with 0.58 μl of 100 mM Flag peptidestock solution (in H₂O) for two hours at 25° C. on a rocking shaker. Thereaction mixture was subsequently dialyzed 2×2 hours against 2 liters of20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C.

C. Coupling of Flag Peptide to Qβ-250 Capsid Protein

A solution of 100 ul of 2 mg/ml Qβ-250 capsid protein in 20 mM Hepes.150 mM NaCl pH 7.2 was reacted for 60 minutes with 7 μl of a solution of65 mM Sulfo-MBS (Pierce) in H₂O at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. 100 μl of the dialyzedreaction mixture was then reacted with 0.58 μl of 100 mM Flag peptidestock solution (in H₂O) for two hours at 25° C. on a rocking shaker. Thereaction mixture was subsequently dialyzed 2×2 hours against 2 liters of20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C.

D. Coupling of Flag Peptides to Qβ-259 Capsid Protein

A solution of 100 ul of 2 mg/ml Qβ-259 capsid protein in 20 mM Hepes.150 mM NaCl pH 7.2 was reacted for 60 minutes with 7 μl of a solution of65 mM Sulfo-MBS (Pierce) in H₂O at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C. 100 μl of the dialyzedreaction mixture was then reacted with 0.58 μl of 100 mM Flag peptidestock solution (in H₂O) for two hours at 25° C. on a rocking shaker. Thereaction mixture was subsequently dialyzed 2×2 hours against 2 liters of20 mM Hepes, 150 mM NaCl, pH 7.2 at 4° C.

The results of the coupling reactions of the Qβ mutants 240, 250 and 259to Flag peptide analyzed by SDS-PAGE are shown in FIG. 1. The loadingpattern was the following:

1. Protein Marker 2. Derivatized Qβ-240 3. Qβ-240 coupled to the Flagpeptide 4. Derivatized Qβ-250 5. Qβ-250 coupled to the Flag peptide 6.Derivatized Qβ-259 7. Qβ-259 coupled to the Flag peptide 8. Derivatizedwt Qβ 9. wt Qβ coupled to the Flag peptide.

Comparison of the derivatized reaction with the coupling reactions showsthat for all the mutants and wt, a coupling band corresponding to 1peptide per subunit is visible. Bands corresponding to 2 peptides persubunit are also visible for the mutant Qβ-250 and wt Q13. The ratio ofthe intensities of the band corresponding to 1 peptide per subunit andto the uncoupled subunit, respectively, is higher for the Qβ-250 mutantand wt Qβ. A weak band corresponding to two peptides per subunit isvisible for the Qβ-240 mutant.

Example 58 Coupling of Flag Peptides to Qβ Mutants Using theCross-Linker SMPH

The Flag peptide, to which a CGG sequence was added N-terminally forcoupling, was chemically synthesized and had the following sequence:CGGDYKDDDDK. This peptide was used for chemical coupling to the Qβmutants as described in the following.

A Coupling of Flag Peptides to Qβ-240 Capsid Protein

A solution of 100 ul of 2 mg/ml Qβ-240 capsid protein in 20 mM Hepes.150 mM NaCl pH 7.4 was reacted for 30 minutes with 2.94 μl of a solutionof 100 mM SMPH (Pierce) in DMSO at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. 90 μl of the dialyzedreaction mixture was then reacted with 1.3 μl of 50 mM Flag peptidestock solution (in DMSO) for two hours at 25° C. on a rocking shaker.The reaction mixture was subsequently dialyzed 2×2 hours against 2liters of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C.

B. Coupling of Flag Peptide to Qβ-250 Capsid Protein

A solution of 100 ul of 2 mg/ml Qβ-250 capsid protein in 20 mM Hepes.150 mM NaCl pH 7.4 was reacted for 30 minutes with 2.94 μl of a solutionof 100 mM SMPH (Pierce) in DMSO at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. 90 μl of the dialyzedreaction mixture was then reacted with 1.3 μl of 50 mM Flag peptidestock solution (in DMSO) for two hours at 25° C. on a rocking shaker.The reaction mixture was subsequently dialyzed 2×2 hours against 2liters of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C.

C. Coupling of Flag Peptide to Qβ-259 Capsid Protein

A solution of 100 ul of 2 mg/ml Qβ-259 capsid protein in 20 mM Hepes.150 mM NaCl pH 7.4 was reacted for 30 minutes with 2.94 μl of a solutionof 100 mM SMPH (Pierce) in DMSO at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. 90 μl of the dialyzedreaction mixture was then reacted with 1.3 μl of 50 mM Flag peptidestock solution (in DMSO) for two hours at 25° C. on a rocking shaker.The reaction mixture was subsequently dialyzed 2×2 hours against 2liters of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C.

The results of the coupling reactions of the Qβ mutants 240, 250 and 259to Flag peptide analyzed by SDS-PAGE are shown in FIG. 1. The loadingpattern was the following. 1. Protein Marker 2. Qβ-240 coupled to Flag,pellet of the coupling reaction 3. Qβ-240 coupled to Flag, Supernatantof the coupling reaction 4. Qβ-240 derivatized with SMPH 5. Qβ-250coupled to Flag, pellet of the coupling reaction 6. Qβ-250 coupled toFlag, supernatant of the coupling reaction 7. Qβ-250 derivatized withSMPH 8. Qβ-259 coupled to Flag, pellet of the coupling reaction 9.Qβ-259 coupled to Flag, supernatant of the coupling reaction 10. Qβ-259derivatized with SMPH.

Comparison of the derivatized reaction with the coupling reactions showsthat for all the mutants, coupling bands corresponding to 1,respectively 2 peptides per subunits are visible. Bands corresponding tothree, respectively four peptides per subunit are also visible for themutant Qβ-250.

Example 59 Coupling of PLA₂-Cys Protein to Mutant Qβ Capsid Proteins

Lyophilized mutant Qβ capsid proteins were swollen overnight in 20 mMHepes, 150 mM NaCl, pH 7.4.

A. Coupling of PLA₂-Cys Protein to Qβ-240 Capsid Protein

A solution of 100 ul of 2 mg/ml Qβ-240 capsid protein in 20 mM Hepes.150 mM NaCl pH 7.4 was reacted for 30 minutes with 2.94 μl of a solutionof 100 mM SMPH (Pierce) in DMSO at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. 90 μl of the dialyzedreaction mixture was mixed with 146 ul 20 mM Hepes, 150 mM NaCl, pH 7.4and reacted with 85.7 ul of 2.1 mg/ml PLA₂-Cys stock solution for fourhours at 25° C. on a rocking shaker. The reaction mixture wassubsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, 150 mMNaCl, pH 7.4 at 4° C.

B. Coupling of PLA₂-Cys Protein to Qβ-250 Capsid Protein

A solution of 100 ul of 2 mg/ml Q13-250 capsid protein in 20 mM Hepes.150 mM NaCl pH 7.4 was reacted for 30 minutes with 2.94 μl of a solutionof 100 mM SMPH (Pierce) in DMSO at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. 90 μl of the dialyzedreaction mixture was mixed with 146 ul 20 mM Hepes, 150 mM NaCl, pH 7.4and reacted with 85.7 ul of 2.1 mg/ml PLA₂-Cys stock solution for fourhours at 25° C. on a rocking shaker. The reaction mixture wassubsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, 150 mMNaCl, pH 7.4 at 4° C.

C. Coupling of PLA₂-Cys Protein to Qβ-259 Capsid Protein

A solution of 100 ul of 2 mg/ml Qβ-259 capsid protein in 20 mM Hepes.150 mM NaCl pH 7.4 was reacted for 30 minutes with 2.94 μl of a solutionof 100 mM SMPH (Pierce) in DMSO at 25° C. on a rocking shaker. Thereaction solution was subsequently dialyzed twice for 2 hours against 2L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4° C. 90 μl of the dialyzedreaction mixture was mixed with 146 μl 20 mM Hepes, 150 mM NaCl, pH 7.4and reacted with 85.7 μl of 2.1 mg/ml PLA₂-Cys stock solution for fourhours at 25° C. on a rocking shaker. The reaction mixture wassubsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, 150 mMNaCl, pH 7.4 at 4° C.

The results of the coupling experiment analyzed by SDS-PAGE are shown inFIG. 1. The loading pattern was the following: 1. Protein Marker 2.derivatized Qβ-240 3. Qβ-240 coupled to Pla2Cys, supernatant of thecoupling reaction 4. Qβ-240 coupled to PLA₂-Cys, pellet of the couplingreaction 5. derivatized Qβ-250 6. Qβ-250 coupled to PLA₂-Cys,supernatant of the coupling reaction 7. Qβ-250 coupled to PLA₂-Cys,pellet of the coupling reaction 8. derivatized Qβ-259 9. Qβ-259 coupledto PLA₂-Cys, supernatant of the coupling reaction 10. Qβ-259 coupled toPLA₂-Cys, pellet of the coupling reaction 11. PLA₂-Cys.

Coupling bands (indicated by the arrow in the figure) were visible forall the mutants, showing that PLA₂-Cys protein could be coupled to allof the mutant Qβ capsid proteins.

1 All patents and publications referred to herein are expresslyincorporated by reference.2 The entire disclosure of U.S. application Ser. No. 09/449,631 and WO00/3227, both filed Nov. 30, 1999, are herein incorporated by referencein their entirety. All publications and patents mentioned hereinaboveare hereby incorporated in their entireties by reference.

What is claimed is:
 1. A composition comprising: (a) a non-naturalmolecular scaffold comprising: (i) a core particle selected from thegroup consisting of: (1) a core particle of non-natural origin; and (2)a core particle of natural origin; and (ii) an organizer comprising atleast one first attachment site, wherein said organizer is connected tosaid core particle by at least one covalent bond, (b) an antigen orantigenic determinant with at least one second attachment site, whereinsaid antigen or antigenic determinant is amyloid beta peptide (Aβ₁₋₄₂)or a fragment thereof, and wherein said second attachment site beingselected from the group consisting of: (i) an attachment site notnaturally occurring with said antigen or antigenic determinant; and (ii)an attachment site naturally occurring with said antigen or antigenicdeterminant, wherein said second attachment site is capable ofassociation through at least one non-peptide bond to said firstattachment site; and wherein said antigen or antigenic determinant andsaid scaffold interact through said association to form an ordered andrepetitive antigen array.
 2. The composition of claim 1, wherein saidassociation is by way of at least one covalent bond.
 3. The compositionof claim 2, wherein said one covalent bond is a non-peptide bond.
 4. Thecomposition of claim 2, wherein said one covalent bond is a peptidebond.
 5. The composition of claim 1, wherein said core particle isselected from the group consisting of: i) a virus; ii) a virus-likeparticle; iii) a bacteriophage; iv) a bacterial pilus; v) a viral capsidparticle; and vi) a recombinant form of (i), (ii), (iii), (iv) or (v).6. The composition of claim 5, wherein said organizer is a polypeptideor residue thereof and said second attachment site is a polypeptide orresidue thereof.
 7. The composition of claim 1 or claim 5, wherein saidcore particle is a virus-like particle.
 8. The composition of claim 7,wherein said virus-like particle is a dimer or multimer of a polypeptidecomprising amino acids 1-147 of SEQ ID NO:158.
 9. The composition ofclaim 8, wherein said virus-like particle is a dimer or multimer of apolypeptide comprising amino acids 1-152 of SEQ ID NO:158.
 10. Thecomposition of claim 9, wherein said first attachment site comprises oris an amino group and said second attachment site comprises or is asulfhydryl group.
 11. The composition of claim 7, wherein saidvirus-like particle is a Hepatitis B virus capsid protein.
 12. Thecomposition of claim 11, wherein said first attachment site comprises oris a lysine residue and said second attachment site comprises or is acysteine residue.
 13. The composition of claim 12, wherein one or morecysteine residues of said Hepatitis B virus capsid protein have beeneither deleted or substituted with another amino acid residue.
 14. Thecomposition of claim 12, wherein said Hepatitis B virus capsid proteincomprises an amino acid sequence selected from the group consisting of:a) the amino acid sequence of SEQ ID NO:89; b) the amino acid sequenceof SEQ ID NO:90; c) the amino acid sequence of SEQ ID NO:93; d) theamino acid sequence of SEQ ID NO:98; e) the amino acid sequence of SEQID NO:99; f) the amino acid sequence of SEQ ID NO:102; g) the amino acidsequence of SEQ ID NO:104; h) the amino acid sequence of SEQ ID NO:105;i) the amino acid sequence of SEQ ID NO:106; j) the amino acid sequenceof SEQ ID NO:119; k) the amino acid sequence of SEQ ID NO:120; l) theamino acid sequence of SEQ ID NO:123; m) the amino acid sequence of SEQID NO:125; n) the amino acid sequence of SEQ ID NO:131; o) the aminoacid sequence of SEQ ID NO:132; p) the amino acid sequence of SEQ IDNO:134; q) the amino acid sequence of SEQ ID NO:157; and r) the aminoacid sequence of SEQ ID NO:158.
 15. The composition of claim 14, whereinone or more cysteine residues of said Hepatitis B virus capsid proteinhave been either deleted or substituted with another amino acid residue.16. The composition of claim 15, wherein the cysteine residuescorresponding to amino acids 48 and 107 in SEQ ID NO:134 have beeneither deleted or substituted with another amino acid residue.
 17. Thecomposition of claim 14, wherein one or more lysine residue of saidHepatitis B virus capsid protein have been either deleted or substitutedwith another amino acid residue.
 18. The composition of claim 1, whereinsaid core particle is a bacterial pilus.
 19. The composition of claim18, wherein said bacterial pilus is a Type-1 pilus of Escherichia coli.20. The composition of claim 19, wherein pilin subunits of said Type-1pilus comprises the amino acid sequence shown in SEQ ID NO:146.
 21. Thecomposition of claim 1, wherein said core particle comprises a bacterialpilin polypeptide.
 22. The composition of claim 21, wherein saidbacterial pilin polypeptide comprises the amino acid sequence shown inSEQ ID NO:146.
 23. The composition of claim 7, wherein said virus-likeparticle comprising recombinant proteins, or fragments thereof, beingselected from the group consisting of: (a) recombinant proteins ofHepatitis B virus; (b) recombinant proteins of measles virus; (c)recombinant proteins of Sindbis virus; (d) recombinant proteins ofRotavirus; (e) recombinant proteins of Foot-and-Mouth-Disease virus; (f)recombinant proteins of Retrovirus; (g) recombinant proteins of Norwalkvirus; (h) recombinant proteins of Alphavirus; (i) recombinant proteinsof human Papilloma virus; (j) recombinant proteins of Polyoma virus; (k)recombinant proteins of bacteriophages; and (l) recombinant proteins ofRNA-phages; (m) recombinant proteins of Qβ-phage; (n) recombinantproteins of GA-phage (o) recombinant proteins of fr-phage; and (p)recombinant proteins of Ty.
 24. The composition of claim 7, wherein saidvirus-like particle comprising, or alternatively essentially consistingof, recombinant proteins, or fragments thereof, of a RNA-phage.
 25. Thecomposition of claim 7, wherein said virus-like particle comprising, oralternatively essentially consisting of, recombinant proteins, orfragments thereof, of a RNA-phage being selected from the groupconsisting of: a) bacteriophage Qβ; b) bacteriophage R17; c)bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f)bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i)bacteriophage NL95; k) bacteriophage f2; and l) bacteriophage PP7. 26.The composition of claim 7, wherein said virus-like particle comprising,or alternatively essentially consisting of, recombinant proteins, orfragments thereof, of bacteriophage Qβ
 27. The composition of claim 7,wherein said virus-like particle comprising, or alternativelyessentially consisting of, recombinant proteins, or fragments thereof,of bacteriophage fr.
 28. The composition of claim 1, wherein said coreparticle is selected from the group consisting of: i) a virus-likeparticle; ii) a bacterial pilus; and iii) a virus-like particle of aRNA-phage.
 29. The composition of claim 7, 11, 14, 18, 24-27, whereinsaid second attachment site does not naturally occur within said antigenor antigenic determinant.
 30. The composition of claim 29, wherein saidcomposition comprises an amino acid linker.
 31. The composition of claim30, wherein said amino acid linker is bound to said antigen or saidantigenic determinant by way of at least one covalent bond.
 32. Thecomposition of claim 31, wherein said covalent bond is a peptide bond.33. The composition of 30, wherein said amino acid linker comprises, oralternatively consist of, said second attachment site.
 34. Thecomposition of claim 33, wherein said amino acid linker comprises asulfhydryl group or a cysteine residue.
 35. The composition of claim 33,wherein said amino acid linker is selected from the group consisting of:(a) CGG (b) N-terminal gamma 1-linker; (c) N-terminal gamma 3-linker;(d) Ig hinge regions; (e) N-terminal glycine linkers; (f)(G)_(k)C(G)_(n) with n=0-12 and k=0-5; (g) N-terminal glycine-serinelinkers (h) (G)_(k)C(G)_(m)(S)_(l)(GGGGS)_(n) with n=0-3, k=0-5, m=0-10,l=0-2; (i) GGC (k) GGC-NH2 (l) C-terminal gamma 1-linker (m) C-terminalgamma 3-linker (n) C-terminal glycine linkers (o) (G)_(n)C(G)_(k) withn=0-12 and k=0-5; (p) C-terminal glycine-serine linkers (q)(G)_(m)(S)_(l)(GGGGS)_(n)(G)_(o)C(G)_(k) with n=0-3, k=0-5, m=0-10,1=0-2, and o=0-8.
 36. The composition of claim 1, wherein said amyloidbeta peptide (Aβ₁₋₄₂) or a fragment thereof is selected from the groupconsisting of: a) Aβ 1-15; b) Aβ 1-27; c) Aβ 1-40; d) Aβ 1-42; e) Aβ33-40; and e) Aβ 33-42.
 37. The composition of claim 36 furthercomprising a heterobifunctional cross-linker, preferably selected fromthe group consisting of: a) SMPH; b) Sulfo-MBS; c) Sulfo-GMBS
 38. Thecomposition of claim 1, wherein said amyloid beta peptide (Aβ₁₋₄₂) orfragment thereof with said second attachment site has an amino acidsequence selected from the group consisting of: a) the amino acidsequence of DAEFRHDSGYEVHHQGGC; b) the amino acid sequence ofCGHGNKSGLMVGGVVIA; and c) the amino acid sequence ofDAEFRHDSGYEVHHQKLVFFAEDVGSNGGC.
 39. The composition of claim 38, whereinsaid core particle is selected from the group consisting of: a) avirus-like particle comprising, alternatively consisting of, recombinantproteins, or fragments thereof of bacteriophage Qβ; b) a virus-likeparticle comprising, alternatively consisting of, recombinant proteins,or fragments thereof of bacteriophage fr; c) a virus-like particle ofHBcAg-lys-2cys-Mut; d) a bacterial pilus; and e) a Type-1 pilus ofEscherichia coli.
 40. The composition of claim 36, wherein said firstattachment site comprises or is an amino group and said secondattachment site comprises or is a sulfhydryl group.
 41. The compositionof claim 36, wherein said first attachment site comprises or is a lysineresidue and said second attachment site comprises or is a cysteineresidue.
 42. The composition of claim 36, wherein said second attachmentsite does not naturally occur within said antigen or antigenicdeterminant.
 43. The composition of claim 42, wherein said compositioncomprises an amino acid linker.
 44. The composition of claim 43, whereinsaid amino acid linker is bound to said antigen or said antigenicdeterminant by way of at least one covalent bond.
 45. The composition ofclaim 43, wherein said covalent bond is a peptide bond.
 46. Thecomposition of 43, wherein said amino acid linker comprises, oralternatively consist of, said second attachment site.
 47. Thecomposition of claim 46, wherein said amino acid linker comprises asulfhydryl group or a cysteine residue.
 48. The composition of claim 36or 46, wherein said amino acid linker is selected from the groupconsisting of: (a) CGG (b) N-terminal gamma 1-linker; (c) N-terminalgamma 3-linker; (d) Ig hinge regions; (e) N-terminal glycine linkers;(f) (G)_(k)C(G)_(n) with n=0-12 and k=0-5; (g) N-terminal glycine-serinelinkers (h) (G)_(k)C(G)_(m)(S)_(l)(GGGGS)_(n) with n=0-3, k=0-5, m=0-10,l=0-2; (i) GGC (k) GGC-NH2, GGC-NMe, GGC-N(Me)2, GGC-NHET or GGC-N(Et)2;(l) C-terminal gamma 1-linker (m) C-terminal gamma 3-linker (n)C-terminal glycine linkers (o) (G)_(n)C(G)_(k) with n=0-12 and k=0-5;(p) C-terminal glycine-serine linkers (q)(G)_(m)(S)_(l)(GGGGS)_(n)(G)_(o)C(G)_(k) with n=0-3, k=0-5, m=0-10,l=0-2, and o=0-8.
 49. The composition of claim 36, wherein said aminoacid linker is selected from the group consisting of: (a) CGG (b) CGKR;(c) CGHGNKS; (d) GGC; (e) GGC-NH2;
 50. A pharmaceutical compositioncomprising: a) the composition of claim 1; and b) an acceptablepharmaceutical carrier.
 51. A method of immunization comprisingadministering the composition of claim 1 to a subject.
 52. A vaccinecomposition comprising the composition of claim
 1. 53. A process forproducing a non-naturally occurring, ordered and repetitive antigenarray comprising: a) providing a non-natural molecular scaffoldcomprising: (i) a core particle selected from the group consisting of:(1) a core particle of non-natural origin; and (2) a core particle ofnatural origin; and (ii) an organizer comprising at least one firstattachment site, wherein said organizer is connected to said coreparticle by at least one covalent bond; and b) providing an antigen orantigenic determinant with at least one second attachment site, whereinsaid antigen or antigenic determinant is amyloid beta peptide (Aβ₁₋₄₂)or a fragment thereof, and wherein said second attachment site beingselected from the group consisting of: (i) an attachment site notnaturally occurring with said antigen or antigenic determinant; and (ii)an attachment site naturally occurring with said antigen or antigenicdeterminant, wherein said second attachment site is capable ofassociation through at least one non-peptide bond to said firstattachment site; and c) combining said non-natural molecular scaffoldand said antigen or antigenic determinant, wherein said antigen orantigenic determinant and said scaffold interact through saidassociation to form an ordered and repetitive antigen array.